Keratin K1 expression vectors and methods of use

ABSTRACT

A keratin K1 vector for expression of a nucleic acid sequence in an epidermal cell. The vector includes a 5&#39; flanking region which includes necessary sequences for expression of a nucleic acid cassette, a keratin K1 3&#39; flanking region which regulates expression of a nucleic acid sequence, predominantly in the epidermis, and a linker which connects the 5&#39; flanking region to a nucleic acid. The linker has a position for inserting a nucleic acid cassette. The linker does not contain the coding sequence of a gene that the linker is naturally associated with. That is, the linker is not the normal gene associated with the 5&#39; and 3&#39; regions.

The invention was partially supported by a grant from the United States Government under HD25479, AI30283 and CA52607 awarded by the National Institutes of Health. Further, this work was partially performed at the National Institutes of Health in the Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute. The U.S. government may have rights in the invention.

RELATED APPLICATION

This application is a continuation-in-part of U.S. patent application Ser. No. 08/145,387 (now abandoned), filed Oct. 29, 1993, entitled "Keratin K1 Expression Vectors and Methods of Use", by Roop et al. (Lyon & Lyon Docket No. 204/144), which is a continuation-in-part of U.S. patent application Ser. No. 07/876,289 (now abandoned), filed Apr. 30, 1992, entitled "Development of a Vector to Target Gene Expression to the Epidermis of Transgenic Animals", by Roop et al. (Lyon & Lyon Docket No. 204/021), both of which are incorporated herein by reference in their entirety, including any drawings.

BACKGROUND OF THE INVENTION

This invention relates to gene therapy to cause expression of genes within the epidermis or epidermal cells.

The skin is the largest organ in the human body. The skin consists of two layers, the epidermis and the dermis. The outer layer is the epidermis which is composed of four histologically defined layers, each of which represent a distinct stage of differentiation of the epidermal keratinocyte. The innermost layer is the stratum germinativum (or basal layer) consisting of continuously dividing cells. The next two layers are the stratum spinosum (or spinous layer) and the stratum granulosum (or granular layer). The outermost layer is the stratum corneum consisting of dead cells whose cytoplasm has been entirely replaced by keratin. (Iverson, et al., Cell Tissue Kinet., Vol. 1, pp. 351-367 (1968); MacKenzie, et al., Nature, Vol. 226, pp. 653-655 (1970)).

The dermis lies under the epidermis and is separated from it by a basement membrane. The dermis is a thick layer of living tissue consisting mainly of a loose connective tissue within which are blood capillaries, lymph vessels, sensory nerve endings, sweat glands and their ducts, hair follicles, sebaceous gland and smooth muscle fibers.

The epidermis is a continuously regenerating epithelium. Keratinocytes are the major cell type of the epidermis and arise from the basal cells in the basal layer. The basal cells consist of metabolically active cells. The basal cells are the cells which undergo mitosis. (Potten, In Stem Cells: Their Identification and Characterization, pp. 200-232 (1983)).

Upon commitment to differentiation, the basal cells lose their proliferative potential and migrate to the spinous layer. With further maturation these cells enter the granular layer and finally terminate as cornified squames in the stratum corneum before being sloughed into the environment. (Matoltsky, J. Invest. Dermatol., Vol. 65, pp. 127-142 (1975)).

During the regeneration process for the differentiated epidermal cells, the cells express a succession of different homologous keratin genes. The keratin produced by the differentiating cells is an insoluble fibrous protein. Keratins are the most abundant proteins synthesized in the epidermal cells and changes in the keratin expression patterns occur during differentiation.

The degree of differentiation can be defined biochemically by the expression of marker proteins that characterize each stage. (Matoltsky, J. Invest. Dermatol., Vol. 65, pp. 127-42 (1975)). For instance, basal keratinocytes express keratins K5 and K14 as major products. (Woodcock-Mitchell, et al., J. Cell Biol., Vol. 95, pp. 580-88 (1982)). These proteins assemble into 10 nm filaments and together with microtubules and microfilaments, comprise the cytoskeleton of epidermal cells. (Steinert, P. M., et al., Cell, Vol. 42, pp. 411-19 (1985)).

One of the earliest changes associated with the commitment to differentiation and migration into the spinous layer is the induction of another differentiation-specific pair of keratins, K1 and K10. Once a cell is committed to the differentiation pathway the cells downregulate the genes for K5 and K14, and express the genes for the differentiation-specific keratins, K1 and K10. (Woodcock-Mitchell, et al., J. Cell Biol., Vol. 95, pp. 580-88 (1982); Roop, et al., Proc. Natl. Acad. Sci., USA, Vol. 80, pp. 716-20 (1983); Schweizer, et al., Cell, Vol. 37, pp. 159-170 (1984)). Transcription of K1 and K10 is restricted to the spinous layer cells. The expression of K1 precedes K10 and is one of the earliest in keratinocyte differentiation. Occasionally, K1 can be observed in the occasional basal cell that has already ceased mitotic activity and is about to migrate into the spinous layer. (Huitfeld, et al., Carcinogenesis, Vol. 12, pp. 2063-2067 (1991)). When the cells mature into granular layer cells, the genes for K1 and K10 are downregulated. At this point, other genes, notably loricrin and filaggrin, are induced. (Dale, B. A., et al., Nature, Vol. 276, pp. 729-731 (1978); Harding, C. R., et al., J. Mol. Biol., Vol. 170, pp. 651-673 (1983)).

Genes or cDNAs encoding the major keratins expressed in epidermal cells have been cloned, such as K5 (Lersch, et al., Mol. and Cell Biol., Vol. 8, pp. 486-493 (1988)), K14 (Marchuk, et al., Proc. Natl. Acad. Sci., USA, Vol. 82, pp. 1609-1613 (1985); Knapp, et al., J. Biol. Chem., Vol. 262, pp. 938-945 (1987); Roop, et al., Cancer Res., Vol. 48, pp. 3245-3252 (1988)), K1 (Steinert, et al., J. Biol. Chem., Vol. 260, pp. 7142-7149 (1985)), and K10 (Krieg, et al., J. Biol. Chem., Vol. 260, pp. 5867-5870 (1985)). In addition, human K6 cDNA has been cloned. (Tyner, et al., Proc. Natl. Acad. Sci., USA, Vol. 82, pp. 4683-4687 (1985)).

Northern blot analysis and in situ hybridization studies suggest that keratin genes K5 and K14 are predominantly transcribed in the proliferating basal layer. Transcription of keratin genes K1 and K10 is induced as cells migrate into the spinous layer. (Lersch, et al., Mol. and Cell Biol., Vol. 8, pp. 486-493 (1988); Knapp, et al., J. Biol. Chem., Vol. 262, pp. 938-945 (1987); Roop, et al., Cancer Res., Vol. 48, pp. 3245-3252 (1988)). K6 is expressed in human skin under conditions of high proliferation and malignant transformation. (Tyner, et al., J. Cell Biol., Vol. 103, pp. 1945-1955 (1986)).

Genes encoding rat and mouse filaggrin have also been identified. In situ hybridization experiments confirmed that transcription of this gene is restricted to the granular layer. (Haydock, et al., J. Biol. Chem., Vol. 261, pp. 12520-12525 (1986); Rothnagel, et al., J. Biol. Chem., Vol. 262, pp. 15643-15648 (1987); Fisher, et al., J. Invest. Dermatol., Vol. 88, pp. 661-664 (1987)).

Loricrin, one of the genes encoding a component of a cornified envelope, has been studied at the molecular level by in situ hybridization showing that transcripts of this gene are restricted to the granular layer. (Mehrel, et al., Cell, Vol. 61, pp. 1103-1112 (1990)). Both the human loricrin gene (Yoneda, et al., J. Biol. Chem., Vol. 267, no. 25, pp. 18060-18066 (1992)), and the mouse loricrin cDNA (Mehrel, et al., Cell, Vol. 61, pp. 1103-1112 (1990)) have been isolated and cloned.

Studies have shown that cells generated by cultivation of a small biopsy can be prepared as stratified sheets and then used for replacement of damaged skin by grafting techniques. (Lindahl, et al., Growth Factors in Health and Disease, p. 388 (1990)). Other studies describe genetically engineered keratinocytes which synthesize human growth hormone. (Morgan, et al., Science, Vol. 237, pp. 1476-1479 (1987)). These studies described retrovirus mediated gene transfer to introduce recombinant human growth hormone into cultured human keratinocytes. The retroviruses were generated from the Ψ AM cell line using an SV40 promoter. (Morgan, et al., Science, Vol. 237, pp. 1476-1479 (1987); Teumer, et al., Growth Hormone and Athymic Mice, FASEB, Vol. 4, pp. 3245-3250 (1990)). The transduced keratinocyte cultures secreted human growth hormone.

In addition, other studies have shown human keratinocytes permanently transformed with plasmids containing the human growth hormone gene under the control of either the metallothionein promoter or the herpesvirus thymidine kinase promoter. (Lindahl, et al., Growth Factors in Health and Disease, p. 388 (1990)). These studies also described skin grafting techniques with the genetically engineered keratinocytes.

SUMMARY OF THE INVENTION

Applicant has determined that it is useful to construct vectors based upon the control sequences of the epidermal-specific gene termed K1. Specifically, expression of such keratin K1 vectors is tissue and differentiation-specific. Keratin K1 vectors can be used to treat diseases by targeting the vector accordingly. These vectors can also be used to create transgenic animals for assessing human disease in an animal model.

The keratin K1 gene is expressed in the epidermis in a differentiation-specific manner. The regulatory elements of such a keratin gene is useful for tissue and differentiation-specific target vectors. Keratin K1 is associated with early differentiation. Occasionally K1 can be observed in the occasional basal cell that has already ceased mitotic activity and is about to migrate into the spinous layer. By using the regulatory elements of this gene, specific expression vectors can be constructed to target the expression of particular nucleic acids in a tissue and differentiation-specific manner.

The 5' regulatory regions of four human epidermal keratin genes, K5, K6, K10 and K14, have been cloned into vectors to drive expression of the CAT reporter gene. These constructs were transfected into epithelial cells along with vectors expressing nuclear receptors for retinoic acid and thyroid hormone. (Tomic, et al., Cell Reg., Vol. 1, pp. 965-973 (1990)). This study demonstrated that these receptors can suppress the promoters of keratin genes. Suppression was ligand dependent and was evident in primary cultures of epithelial cells. Other studies have discussed the regulation by calcium of human keratin genes K1 and K10. (Rosenthal, et al., Cell Growth and Differentiation, Vol. 2, pp. 107-113 (1991)). 5' and 3' flanking sequence for the human K1 gene responded to elevated levels of calcium in order to induce both mouse K1 and human K1 expression.

Furthermore, both the 5' and 3' sequences for human K1 keratin gene have been used to express oncogenes exclusively to the epidermis of transgenic mice. (Greenhalgh, et al., Mol. Carcinogenesis, Vol. 7, pp. 99-110 (1993); Greenhalgh, et al., Oncogene, Vol. 8, pp. 2145-2157 (1993)). The control elements of 5' and 3' flanking sequences of the human keratin K1 gene that respond to calcium and differentiation were studied by mutations to the 5' and 3' sequences. These studies further define DNA regulatory elements for calcium induced differentiation responses. (Huff, et al., J. Biol. Chem., Vol. 268, No. 1, pp. 377-384 (1993)).

Other expression vectors have been constructed with the K1 5' and 3' sequences to target TGF-β to the epidermis. This study involved the role of TGF-β as an inhibitor of epithelial-cell proliferation. (Sellheyer, et al., PNAS, Vol. 90, pp. 5237-5241 (1993)). In addition, a bovine K6 vector was also constructed to study the role of TGF-β in hair follicles. (Blessing, et al., Genes & Dev., Vol. 7, pp. 204-215 (1993)).

Taking advantage of the unique targeting ability of epidermal cells, the present invention features use of the keratin K1 gene regulatory regions to construct vectors which direct efficient expression of exogenous DNA in epidermal cells. In particular, the present invention demonstrates that by removing sequences that normally restrict expression of the keratin K1 gene in early differentiated cells, an expression vector can be constructed which achieves high levels of expression in undifferentiated epidermal cells. Such expression is greater than equivalent vectors which use the viral promoter SV40. The vector can be constituitively expressed in epidermal cells at all differentiation states, not just the spinous layer. Likewise, if the sequences that normally restrict expression of the K1 gene are not removed, an expression vector may be constructed which directs expression of exogenous DNA to only cells of the earlier differentiated layers of the epidermis.

This unique targeting ability also allows transgenic animal models used for not only the dissection of molecular carcinogenesis and disease, but also in assessing potential chemical and physical carcinogens and tumor promoters, and exploring novel therapeutic avenues. Furthermore, advantages due to the unique targeting ability of the above vector allow methods to administer and treat wounds, surgical incisions, skin ulcers, psoriasis and cancer. Furthermore, the above vectors can be used to transform epidermal cells to produce particular proteins, polypeptides, and RNA, as well as be used in methods for creating immune responses.

Likewise, the above expression vector can be used in vitro with epidermal cells in culture. Use of this vector in vitro allows the role of various nucleic acids to be studied by target specific expression into epidermal cells. (Greenhalgh, et al., Mol. Carcinogenesis, Vol. 7, pp. 99-110 (1993); Greenhalgh, et al., Oncogene, Vol. 8, pp. 2145-2157 (1993)).

It should also be noted that this invention features vectors using the regulatory elements required for specific nucleic acid expression in epidermal cells, including regulatory elements from the K1 gene, as well as other regulatory elements of specific genes which are not K1.

In the first aspect, the present invention features a keratin K1 vector for expression of a nucleic acid sequence in an epidermal cell. The vector includes a 5' flanking region which includes necessary sequences for expression of a nucleic acid cassette, a keratin K1 3' flanking region which regulates expression of a nucleic acid sequence, predominantly in the epidermis, and a linker which connects the 5' flanking region to a nucleic acid. The linker has a position for inserting a nucleic acid cassette. The linker does not contain the coding sequence of a gene that the linker is naturally associated with. That is, the linker is not the normal gene associated with the 5' and 3' regions.

The term "vector" as used herein refers to a nucleic acid, e.g., DNA derived from a plasmid, cosmid, phasmid or bacteriophage, into which fragments of nucleic acid may be inserted or cloned. The vector can contain one or more unique restriction sites for this purpose, and may be capable of autonomous replication in a defined host or organism such that the cloned sequence is reproduced. The vector molecule can confer some well-defined phenotype on the host organism which is either selectable or readily detected. Some components of a vector may be a DNA molecule incorporating DNA, a sequence encoding a therapeutic or desired product, and regulatory elements for transcription, translation, RNA stability and replication. A viral vector in this sense is one that contains a portion of a viral genome, e.g. a packaging signal, and is not merely DNA or a located gene within a viral particle.

The purpose of the vector is for expression of a nucleic acid sequence in an epidermal cell. Expression includes the efficient transcription of an inserted gene or nucleic acid sequence within the vector. Expression products may be proteins, polypeptides or RNA. The gene insert or nucleic acid sequence is contained in the nucleic acid cassette.

The term "nucleic acid cassette" as used herein refers to the genetic material of interest which can express a protein, polypeptide or RNA. The nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein or a polypeptide in the transformed epidermal cell. Preferably, the cassette has its 3' and 5' ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end.

A variety of proteins and polypeptides can be encoded by the sequence in a nucleic acid cassette in the transformed epidermal cells. Those proteins or polypeptides which can be expressed include hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressor, viral antigens, parasitic antigens and bacterial antigens. Specific examples of these compounds include proinsulin, insulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding protein, epidermal growth factor TGF-α, TGF-β, dermal growth factor (PDGF), angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin), matrix proteins (Type IV collagen, Type VII collagen, laminin), oncogenes (ras, fos, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p53 protein, cytokine receptor, IL-1, IL-6, IL-8, viral capsid protein and proteins from viral, bacterial and parasitic organisms which can be used to induce immune responses. In addition, the nucleic acid cassette can code for antisense RNA or ribosomes as well. These are only examples and are not meant to be limiting in any manner.

In addition, the nucleic acid cassette can encode a "transforming gene" which encompasses viral oncogenes, endogenous proto-oncogenes and activated proto-oncogenes. A variety of oncogenes are known in the art. The term "oncogene" means those genes which cause cancer and include both viral and cellular oncogenes, many of which are homologous to DNA sequences endogenous to rodents and/or humans. The term "oncogene" includes both the viral sequence and the homologous endogenous sequences. Some examples of transforming genes are Ha-ras, Ki-ras, N-ras, fos, myc, src, sis, erbA, erbB, jun, p Large T, p Middle T, HPV E7, HPV E6, HPV E5, tat, EIA, Rb, p53, WT1, TGF-α, TGF-β, EGFR, RAR, VD₃ R, and PKC.

The term "flanking region" as used herein refers to nucleotide sequences on either side of an associated gene. Flanking regions can be either 3' or 5' to a particular gene in question. In general, flanking sequences contain elements necessary for regulation of expression of a particular gene. This can include regulatory sequences necessary for tissue-specific expression, differentiation-specific expression, as well as sequences necessary for efficient expression.

Usually, specific regulatory sequences or elements are embedded adjacent to or within the protein coding regions of DNA. These elements, located adjacent to the gene, are termed cis-acting elements. The signals are recognized by other diffusible biomolecules in trans to potentiate the transcriptional activity. These biomolecules are termed "trans-acting factors". The presence of the trans-acting factors and cis-acting elements have been shown to contribute to the timing and developmental expression pattern of a gene. Cis-acting elements are usually thought of as those that regulate transcription and are found within promoter regions and other upstream (5') or downstream (3') DNA flanking regions.

Flanking DNA with regulatory elements that regulate expression of the genes of the epidermis may also include modulator sequences that are regulated by specific factors, such as Vitamin D and its metabolites, Vitamin A and its metabolites, retinoic acid, and calcium, as well as others. "Modulator Sequences" as used herein refers to sequences which may be in the 3' or 5' flanking regions where such sequences can enhance activation and/or suppression of the transcription of the associated gene. "Responsive" or "respond" as used herein in relation to modulate relates to the enhancement of activation and/or suppression of gene transcription as discussed below. "Metabolites" as used herein refers to any product of metabolism.

The 5' flanking regions may include a promoter, a TATA box, a CAP site and a first intron and intron/exon boundary which are in an appropriate relationship sequentially and positionally for the expression of an associated gene. In this invention, "necessary sequences" are those elements of the 5' flanking region which are sequentially and positionally in an appropriate relationship to cause the specific expression of a nucleic acid cassette. The 5' flanking region can provide tissue-specific expression to an associated gene.

The 5' sequence may contain elements which regulate tissue-specific expression. The 5' flanking region is located 5' to the associated gene or nucleic acid sequence to be expressed. The 5' flanking region regulatory elements can include the portion of a naturally occurring 5' element responsible for tissue-specific expression. The 5' flanking region can be defined by known procedures. For example, the active portion of the 5' flanking region can be mapped by mutational analysis or various clones created to define the desired activity in a selected vector.

The term "promoter" as used herein refers to a recognition site of a DNA strand to which the RNA polymerase binds. The promoter usually is a DNA fragment of about 100 to 200 base pairs (in eucaryotic genes) in the 5' flanking DNA upstream of the CAP site or the transcriptional initiation start site. The promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity. The complex can be modified by activating sequences termed "enhancers" or inhibitory sequences termed "silencers". The promoter can be one which is naturally or non-naturally associated with a 5' flanking region.

The term "intron" as used herein refers to a section of DNA occurring in a portion of a gene which does not code for an amino acid in the gene product. RNA transcribed from such an intron is included in a precursor RNA, from which the intron mRNA is then excised (and is therefore not transcribed into messenger RNA nor translated into protein).

The term "exon" as used herein refers to a portion of a gene that is included in the transcript of a gene and survives processing of the RNA in the cell to become part of a messenger RNA. Exons generally occupy three distinct regions of genes that encode proteins. The first, which is not translated into proteins, signals the beginning of RNA transcription and contains sequences that direct the messenger RNA to the ribosomes for protein synthesis. The exons in the second region contain the information that is translated into the amino acid sequence of the protein Exons in the third region are transcribed into the part of the messenger RNA that contains the signals for termination of translation and for the addition of polyadenylation tail (poly(A)).

The intron/exon boundary will be that portion in a particular gene where an intron section connects to an exon position. The terms "TATA box" and "CAP site" are used as they are recognized in the art.

The 3' flanking region contains sequences which regulate expression predominantly in the epidermal cells of a nucleic acid sequence. The 3' flanking regions provide tissue-specific expression to an associated gene. The 3' flanking region may be located within a vector of this invention either 5' or 3' to that of an associated gene in order to regulate its expression. The term as used herein includes that portion of the naturally occurring 3' flanking region responsible for tissue-specific expression. That portion can be readily defined by known procedures. For example, the active portions of a 3' flanking region can be mapped by mutational analysis or various clones created to define the desired activity in a selected vector system.

The 3' flanking region may also contain a 3' untranslated region or 3' UTR. This term refers to the sequence at the 3' end of a structural gene which is usually transcribed with the gene. This 3' UTR region usually contains a poly(A) sequence. Although the 3' UTR is transcribed from the DNA, it is not translated into protein. Keratin-specific 3' UTR sequences may be used to allow for specific stability in a keratinocyte or epidermal tissues.

A "3' non-coding region" or "3' NCR" is a region contiguous to the 3' UTR region of a structural gene. The 3' NCR region generally contains a transcriptional termination signal.

The 3' UTR and 3' NCR sequences provide a higher level of messenger RNA accumulation through increased messenger RNA stability in keratinocytes rather than non-keratinocyte cells. Thus, this increased stability of messenger RNA leads to increased levels of protein production. It should also be noted that the 5' flanking region can also contain UTR sequences.

The 3' flanking regions from a keratin K1 gene regulates expression predominantly in the epidermis. "Predominantly" as used herein means that the gene associated with the 3' flanking region, whether natural or in the expression vector, will be expressed to a higher degree only in the epidermis, i.e., to the same order of magnitude of difference as would be found in natural expression of the keratin K1 gene in the epidermis versus other cell types. In addition, the same magnitude of difference may be observed in an epidermis versus other cell types by Northern analysis, X-Gal, immunofluorescence or CAT assays as discussed herein and known in the art. While keratin genes are normally expressed in the epidermis or epidermal cells, namely, keratinocytes, keratin genes are expressed to a lower degree in other tissues, such as the oral mucosa, esophagus and trachea, and other tissues as well. The 3' flanking region as used herein will also express the associated gene in other tissues but to a lower degree than expression in the epidermis. Expression is preferentially in the epidermis.

By "keratin K1 gene" is meant those genes exemplified herein and their equivalents in other animal species or other tissues. Homologous or analogous sequences are also included so long as they provide equivalent regulatory properties to those described herein. It is important in this invention that the chosen sequence provide the tissue specific expression noted herein. In addition, other sequences such as the modulators and regulators noted herein include such analogous sequences and functionalities. Those in the art will recognize that the minimum sequences required for such a function are encompassed by such a definition and are readily determined by standard technique exemplified herein.

In addition to the above, the 3' flanking region may also contain approximately 8.0 kb of a 5' flanking sequence (or the functional sequence therein) from the 18 kb EcoRV fragment. An 18 kb EcoRV fragment is from the human keratin K1 gene and is expressed exactly like the endogenous mouse K1 gene, i.e., post mitotically in cells committed to terminal differentiation. This may be inserted at the end of the vectors at the 3' flanking region. Expression of this vector will only be in cells after they commit to terminal differentiation.

The term "linker" as used herein refers to DNA which contains the recognition site for a specific restriction endonuclease. Linkers may be connected to the ends of DNA fragments prepared by cleavage with some other enzyme. A linker having a unique restriction endonuclease site at the location of the start and stop codon connects the 5' flanking region to a nucleic acid. In particular, the linker provides a position for inserting the nucleic acid cassette which contains a specific nucleic acid sequence to be expressed. This position may be an endonuclease site in the linker, such as Cla I, Not I, Xma I, Bgl II, Pac I, Xho I, Nhe I and Sfi I.

In preferred embodiments, the vector described above may have both its 5' flanking region and its 3' flanking region from keratin K1 gene. In particular, the present invention may have a 5' flanking region of approximately 1.2 kb, and a 3' flanking sequence of approximately 2.1 kb of a keratin K1 gene.

As discussed above, these regions can be further and more precisely defined by routine methodology. Preferably, the vector contains such a 3' region or 5' region comprising, consisting, or consisting essentially of these regions. The term "consisting of" is used herein as it is recognized in the art. A vector with the 3' or 5' regions "consisting essentially of" the regions of the present invention includes those regions in which the sequence is changed, but the desired vector activity remains equivalent. Such a change, for example, could be a change of 10 nucleotides in any of the above regions. This is only an example and is non-limiting.

More particularly, the vector above, may contain a 5' flanking region having nucleotides 1 to 46 of Sequence ID No. 1, a 3' flanking region having nucleotides 6891 to 10747 of Sequence ID No. 1, and a linker having nucleotides 2351 to 2376 of Sequence ID No. 2.

The invention can also feature a vector as described above with 5' UTR sequences, 3' UTR sequences, and 3' NCR sequences. These can be incorporated into the vector to allow the nucleic acid in the cassette to be transcribed into RNA and then when necessary, translated into proteins or polypeptides in the transformed epidermal cell.

A second aspect of the present invention is a purified nucleic acid sequence comprising the keratin K1 gene 5' flanking region and the 3' flanking region of Sequence ID No. 1. "Purified" as used herein means that the sequence is isolated from its natural state. The present invention also covers the 5' flanking region or the 3' flanking region by themselves. Not only does the invention cover either of the 5' flanking region and/or 3' flanking region of the isolated keratin K1 gene, but other equivalent keratin K1 genes as well.

In a third related aspect, the present invention features an epidermal cell transformed with a vector as described above for expression of a nucleic acid sequence. As described above, the nucleic acid cassette may contain genetic material encoding for a variety of proteins, polypeptides or RNA.

As used herein, "transformation" is a mechanism of gene transfer which involves the uptake of DNA by a cell or organism. Following entry into the cell, the transforming DNA may recombine with that of the host or may replicate independently as a plasmid or temperate phage. Cells which are able to take up DNA are described as competent. Particular cells may not be naturally competent, but require various treatments in order to induce the transfer of DNA across the cell membrane.

Transformation can be performed by in vivo techniques as described below, or by ex vivo techniques in which epidermal cells are co-transfected with a vector containing a selectable marker This selectable marker is used to select those cells which have become transformed. It is well known to those skilled in the art the type of selectable markers to be used with transformation studies.

The transformed cell can produce a variety of compounds selected from proteins, polypeptides or RNA, including hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, tumor antigens, viral antigens, parasitic antigens and bacterial antigens. Other examples can be found above in the discussion of nucleic acid cassette. The product expressed by the transformed cell depends on the nucleic acid of the nucleic acid cassette. As discussed above, this list is only an example and is not meant to be limiting.

A fourth aspect of the present invention features methods for transformation of epidermal cells. These methods comprise the steps of contacting a cell with a vector as described above for a sufficient time to transform the epidermal cell.

In a fifth aspect, the present invention features a method for treating a wound or surgical incision. In addition, the present invention features a method to treat skin ulcers. These methods use the above-referenced vectors in order to transform epidermal cells. The nucleic acid cassette of the vector contains genetic material coding for a growth factor, a matrix protein or angiogenesis factor. Expression of such genes in vivo aids in the treatment of wounds or surgical incisions. As above, the methods of transformation can be by in vivo or ex vivo techniques.

In a more particular related aspect, the methods involve transforming epidermal cells with a plurality of the above-referenced vectors. In these particular methods, the genetic material of at least one vector codes for a growth factor, the genetic material of at least one vector codes for a second growth factor, the genetic material of at least one vector codes for a matrix protein and the genetic material of at least one vector codes for an angiogenesis factor. The growth factors may consist of epidermal growth factor, transforming growth factor, dermal growth factor or even growth hormone. The matrix protein may consist of Type IV collagen, laminin, nidogen or Type VII collagen. The angiogenesis factor may consist of acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin.

Transformation in these methods and those below can be performed by in vivo techniques, as well as ex vivo techniques. Ex vivo techniques also can include transplanting the transformed epidermal cells into the animal or human to be treated. Such an ex vivo procedure is used with treating wounds or surgical incisions or skin ulcers and other methods below.

A sixth related aspect of the present invention features a method for treating psoriasis by transforming epidermal cells with the above-referenced vectors. These vectors contain nucleic acid sequences coding for proteins, polypeptides or RNA, such as transforming growth factors or cytokine receptors. The RNA which is produced by the expression vector may be antisense RNA complementary to transforming growth factor alpha, IL-1, IL-6 or IL-8. The cytokine receptors may be receptors for IL-1, IL-6 or IL-8. "Receptor" as used herein includes natural receptors as well as anything that binds a ligand and causes compartmentalization changes in a cell.

A seventh related aspect of the present invention features a method for treating cancer. This method includes the transformation of squamous epithelial cells with the above-referenced vectors. The nucleic acid cassettes of the above vectors contain genetic material coding for proteins, polypeptides or RNA. In particular, the genetic material may code for the p53 protein or code for antisense RNA which is complementary to the E6 or E7 gene of human papilloma virus.

Squamous epithelial cells as used herein, are cells which may be either epidermis cells, oral mucosa, esophageal, vaginal, trachea or corneal epithelia.

An eighth related aspect of the present invention features a method for inducing an immune or immunological response by transforming an epidermal cell with the above-referenced vector. The nucleic acid cassette may contain nucleic acid sequences coding for proteins or polypeptides, or other factors which might produce an immunogenic or immunological response. The nucleic acid cassette can contain genetic material that encodes for microbial proteins. This includes genetic material coding for a viral capsid protein from the human papilloma virus, other viral capsids, bacterial proteins and toxins. This is only an example and is not meant to be limiting.

A ninth related aspect of the invention features a transgenic animal whose cells contain the vector referenced above. These cells include germ or somatic cells. Transgenic animal models can be used for not only dissection of molecular carcinogenesis and disease, but also in assessing potential chemical and physical carcinogens and tumor promoters, and exploring therapeutic avenues.

The genetic material which is incorporated into the epidermal cells from the above vectors includes DNA not normally found in epidermal cells, DNA which is normally found in epidermal cells but not expressed at physiological significant levels, DNA normally found in epidermal cells and normally expressed at physiological desired levels, and other DNA which can be modified for expression in epidermal cells, and any combination of the above.

The vectors of the above methods may be administered by various routes. The term "administration" refers to the route of introduction of a vector or carrier of DNA into the body. Administration may be intravenous, intramuscular, topical, oral, or by gene gun or hypospray instrumentation. Administration can be directly to a target tissue or through systemic delivery. Administration will include a variety of methods, such as direct gene transfer into skin tissue by liposomes, proteoliposomes, calcium phosphate-coprecipitated DNA, DNA coupled to macromolecular complexes, DNA transporters, DNA coded to microprojectiles, coded plasmids, direct microinjection, as well as skin grafts. Direct gene transfer of vectors can be administered by direct microinjection, electroporation, liposomes, proteoliposomes, calcium phosphate-coprecipitation, skin grafts, retroviral vectors, DNA coupled to macromolecular complexes, DNA transporters and microprojectiles. Routes of administration include intramuscular, aerosol, oral, topical, systemic, ocular, intraperitoneal and/or intrathecal. See, e.g., WO 93/18759, hereby incorporated by reference herein.

Other features and advantages of the invention will be apparent from the following detailed description of the invention in conjunction with the accompanying drawings and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the human keratin K1 gene (HK1) and the expression vector derived from its regulatory sequences.

FIG. 2 demonstrates the suppression of the SV40 promoter by a novel negative regulatory element from the HK1 gene (HK1.NRE) in the presence of Vitamin D₃.

FIG. 3 is a schematic drawing of the HK1 vector containing the coding sequence of v-ras^(Ha) protein of Harvey Murine Sarcoma Virus.

FIG. 4 is a schematic drawing of the HK1 vector containing the coding sequence of the v-fos protein from a FBJ/FBR chimeric plasmid.

FIG. 5 is a schematic drawing of the HK1 expression vector containing the coding sequences of the E6 and E7 proteins from human papilloma virus 18.

FIG. 6 is a schematic drawing of the HK1 vector containing the coding sequence of TGF-α.

FIG. 7 is a schematic drawing of the HK1 vector containing the coding sequence of the trans-regulatory protein tat, from human immunodeficiency virus.

FIG. 8 is a schematic drawing of an 18 kb EcoRV fragment containing the HK1 gene.

FIG. 9 is a schematic drawing of a derivative of the HK1 vector containing additional 5' flanking sequences which restrict expression to differentiated epidermal cells.

FIG. 10 illustrates the nucleotide sequence of the protected regions FP(A) and FP(BP). The consensus AP-1 site within FP(A) is underlined. The canonical hormone response elements within FP(B) are denoted by arrows.

The drawings are not necessarily to scale, and certain features of the invention may be exaggerated in scale and shown in schematic form in the interest of clarity and conciseness.

DETAILED DESCRIPTION OF THE INVENTION

It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The following examples are offered by way of illustration and are not intended to limit the invention in any manner.

The following are specific examples of preferred embodiments of the present invention. These examples demonstrate how keratin K1 based vectors can be used in construction of various cellular or animal models, and how genes can be regulated by sequences within such vectors. The utility of such vectors is noted herein and is amplified upon in co-pending application by Roop et al., entitled "Specific Expression Vectors and Methods of Use", supra, and such sections are hereby specifically incorporated by reference herein.

Below are provided examples of specific regions of the keratin K1 genes that can be used to provide certain functionalities to an expression vector, and thus within a transformed cell or animal containing such a vector. Those in the art will recognize that specific portions of these regions can be identified as that containing the functional nucleic acid sequence providing the desirable property, and such regions can be readily minimized using routine deletion or mutagenic techniques or their equivalent. Thus, such regions include the modulator sequence described below, as well as those sequences responsive to calcium, Vitamin D and its metabolite, Vitamin A and its metabolite, and progesterone. As noted herein, such controlling segments of nucleic acid may be inserted at any location on the vector, although there may be preferable sites as described herein.

EXAMPLE 1 Construction and Characterization of a Vector

To target the expression of exogenous DNA to the epidermis a vector from the human keratin K1 gene was constructed. Among its many uses, it is useful in making transgenic animals.

A schematic showing the structure of the human keratin K1 gene is shown in FIG. 1. The 10.8 kb EcoRI fragment containing the entire human keratin K1 gene was originally isolated from lambda clone c55 (Johnson, et al., PNAS, USA, Vol, 82, pp. 1896-1900 (1985)). In constructing the targeting vector, most of the first exon including the ATG was removed, leaving only the 5' non-coding sequences, the first intron and the intron/exon boundaries. In addition, the remainder of the gene up to the termination codon was deleted. A polylinker containing the following unique restriction sites (Bam HI, Xma I, Kpn I, Not I, and Cla I) was engineered into a site 3' of the first intron to allow easy insertion of exogenous DNA. These manipulations were performed through the use of polymerase chain reactions (PCR). The unique EcoRI sites were conserved at the ends of the vector to allow easy amplification in pGEM vectors and excision for purification from plasmid sequences prior to injection into embryos.

The rationale for constructing the vector in this manner was as follows. Since the specific elements responsible for the expression characteristics of the 10.8 kb human keratin K1 fragment are not defined, the entire 5' and 3' flanking regions were included in the vector construct. One skilled in the art will readily recognize that as these elements are further defined the flanking sequences can be changed accordingly. For example, the vector may contain a nucleic acid sequence consisting of the 10.8 kb EcoRI fragment and an additional 8.1 kb of sequence 5' to the 10.8 kb EcoRI fragment, comprising an approximately 18 kb EcoRV nucleic acid sequence. In addition, sequences within the 3' non-coding region were retained since these may confirm stability to transcripts of exogenous DNA in epidermal cells. Further description of 3' regulatory elements are set forth in Example 16. The first intron was retained to potentially enhance expression efficiency (Brinster, et al., PNAS, USA, Vol. 82, pp. 1896-1900 (1988).

EXAMPLE 2 HK1 Expression in Epidermal Keratinocytes

To assess the human keratin K1 targeting vector for exclusive expression in epidermal keratinocytes, the β-galactosidase reporter gene was cloned into Bam HI and Cla I restriction sites located in the polylinker region of the expression vector (FIG. 1). The β-galactosidase gene has frequently been used as a reporter gene to assess targeting specificity (MacGregor, et al., In: Methods in Molecular Biology, Vol. 7, pp. 217-235 (1991). This construct was designated pHK1.β-gal. To determine if expression of this construct resulted in the production of a functional protein, and to determine whether the vector retained cell type specificity, this construct was transfected into primary epidermal keratinocytes and primary dermal fibroblasts. At seventy-two hours post transfection cells were stained with a solution containing the substrate 5-bromo-4-chloro-3-indoyl-β-galactosidase (X-gal). β-galactosidase activity, indicated by a blue coloration, was detected in keratinocytes but not fibroblasts. Thus, expression of the HK1.β-gal construct was cell type specific and resulted in the production of a functional protein.

EXAMPLE 3 Transgene Mice

The same pHK1.β-gal construct utilized in the in vitro studies discussed in Example 2 was used in the production of transgenic mice. This construct was digested with EcoRI (see FIG. 1) and subjected to preparative agarose gel electrophoresis to purify the pHK1.β-gal expression construct away from plasmid sequences (pGEM 3) which might interfere with expression. The separated expression construct sequences were purified and recovered using NA 45 DEAE membrane (Schleicher & Schuell). DNA was precipitated and resuspended at 1-3 ng/μl. ICR outbred female mice (Sasco) were given PMS and HCG to stimulate superovulation, mated to FVB males (Taconic) and the resulting early fertilized embryos (most preferably on cell stage) were collected from the oviducts. DNA was micro-injected into the pronuclei and the embryos were surgically transferred to pseudopregnant recipient females (the result of mating ICR females with vasectomized B₆ D₂ F₁ males (Taconic)).

In the initial experiments, 40 mice were born. In order to quickly determine if the pHK1.β-gal transgene was being exclusively expressed in the epidermis of these mice, these animals were sacrificed at birth. A small amount of tissue was removed for extraction of DNA and the remainder of the neonate was rapidly frozen in Tissue-Tek O.C.T. for frozen sections. PCR analysis was performed on the extracted DNA using oligonucleotide primers specific for the intron within the HK1 vector and this demonstrated that five of the 40 neonates contained the HK1.β-gal construct.

To assess whether expression of the HK1β-gal construct was restricted to the epidermis or expressed in other squamous epithelia, frozen longitudinal sections were cut from several PCR positive and PCR negative embedded neonates and these were stained with X-Gal. Data showed where a PCR positive animal, #30, expressed high levels of β-galactosidase in the epidermis and a PCR negative sibling, #29, was completely negative, indicating that endogenous murine β-galactosidase was not expressed at sufficient levels in the epidermis to cause false positives in this assay. Staining of the intestine was observed in both the positive (#30) and negative (#29) neonates This may represent endogenous enzyme activity or the production of β-galactosidase by bacteria in the intestine. X-gal staining was detected in the basal compartment, although it is not as intense as in the differentiated layers. Thus, the human keratin K1 expression vector is also expressed in a substantial number of proliferating basal cells.

The most important finding from these initial transgenic experiments is that the vector constructed from the human keratin K1 gene can target the expression of an exogenous coding sequence exclusively to the epidermis of transgenic mice. This specificity of targeting was readily shown by the data. A low power exposure of the skin of #30 demonstrates intense staining with X-Gal. In addition, there are numerous hair follicles and sebaceous glands which do not stain with X-Gal. Keratins K5 and K14 are not only expressed in the epidermis, but in all squamous epithelia, including hair follicles and sebaceous glands. The expression pattern for keratin K14 is revealed by immunofluorescence with a specific K14 antiserum. Staining of the epidermis, as well as hair follicles and sebaceous glands is observed. If the strategy used in construction the human keratin K1 expression vector had altered its targeting specificity in transgenic mice, then X-Gal staining would have been observed in hair follicles, sebaceous glands, other squamous epithelia, and perhaps even other tissue types. However, expression of the HK1.β-gal transgene, like the keratin K1 gene itself is restricted to the epidermis

EXAMPLE 4 Regulation of Keratin K1 Vector by Vitamin D₃

A novel Vitamin D₃ responsive element was used to modulate expression levels in the epidermis. Although all of the regulatory elements of the human keratin K1 gene have not been identified, a novel negative regulatory element from the human keratin K1 gene (HK1.NRE) has been identified and this example demonstrates that it is able to suppress a heterologous promoter in response to Vitamin D₃. The HK1.NRE is 70 nucleotides in length (nucleotides 9134 to 9204 of Sequence ID No. 1). PCR technology was used to generate Bam HI and Bgl II sites at opposite ends of this fragment. This facilitates generating multiple copies of this fragment since ligation and digestion with Bam HI and Bgl II will select for oligomers which have ligated head to tail. Four tandem copies of the HK1.NRE were inserted into the Bgl II cloning site of pA10.CAT. In the absence of Vitamin D₃ this construct is highly expressed when transfected into primary mouse epidermal cells (FIG. 2). The addition of increasing concentrations of Vitamin D₃ to the culture medium completely suppresses transcription of this heterologous promoter. This observation indicates that the activity of the human keratin K1 expression vector can be modulated in the epidermis. The activity of the human keratin K1 vector is suppressed in the epidermis by topical application of Vitamin D₃, or an analogue, to the skin. In addition, it was determined that retinoic acid restored activity of the K1 vector. Further discussion of Vitamin D regulation is set forth in Examples 8 and 16.

EXAMPLE 5 Development of Transgenic Animal Models for Skin Carcinogenesis

The ability to stably introduce genes into the germline of mice has greatly enhanced prospects for generation of animal models of human disease (Leder and Stewart, U.S. Pat. No. 4,736,866 issued Apr. 12, 1988, and Palmiter and Brinster, Ann. Rev. Genet., Vol. 20, pp. 465-499). When such genes are combined with regulatory sequences that target their expression to specific tissues, it provides a model to not only study diseases in the context of living organisms, but also in specific tissues suspected of being the targets of these genes. Thus, transgenic mice offer the possibility to determine the influence of factors such as blood supply, an intact immune system, humoral and cell-mediated growth controls and physical barriers on disease progression. The epidermis is an attractive tissue for targeted gene expression; not only is it a model for epithelial diseases in general but the accessibility of the epidermis allows easy detection of progressive pathological changes that result from transgene expression as well as the assessment of the potential role played by environmental factors in these processes. In addition, the prospects for utilizing gene therapy to treat cancer are coming closer to reality. Therefore, animal models of human cancers would be useful to assess the therapeutic potential of these approaches. The development of animal models of skin disease is dependent upon the ability to specifically target gene expression to the epidermis. The human keratin K1 targeting vector described in Example 1 is ideally suited for this purpose.

EXAMPLE 6 Targeting the v-ras^(Ha) Oncogene to the Epidermis

One family of proto-oncogenes, the ras family (ras^(Ha), ras^(K1), ras^(N)), has been identified in approximately 20% of human tumors by virtue of specific point mutations at codons 12, 13, and 61 which activate their transforming potential. The mechanisms whereby ras genes become activated are currently unknown but there is widespread evidence that environmental agents play pivotal roles in the etiology of ras mutations. To date, few studies have undertaken to study ras activation in human skin malignancies. However recent reports have identified ras^(Ha) activation in basal and squamous cell carcinomas appearing on sun exposed body sites, interestingly at potential pyrimidine dimer sites possibly derived from skin exposure to UV irradiation. In the mouse skin model of chemical carcinogenesis where the three distinct stages of initiation, promotion and malignant conversion have been defined, ras^(Ha) activation has been found in benign squamous papillomas, the end point of initiation and promotion suggesting an early role for ras^(Ha) in skin carcinogenesis. Taken collectively, the above experimental evidence suggests the importance of developing an animal model to further study the mechanism of ras^(Ha) -induced skin carcinogenesis. Toward this end, the sequence encoding the v-ras^(Ha) protein of Harvey Murine Sarcoma Virus (Dhar, et al., Science, Vol. 217, pp. 934-937 (1982) was cloned into the Bam HI and Cla I sites of the human keratin K1 expression vector (FIG. 3) To discriminate expression of the v-ras^(Ha) transgene from that of the endogenous ras gene, a sequence encoding the human keratin K6 epitope Sequence ID No. 4 was engineered onto the 5' end of the v-ras^(Ha) cassette.

HK1 ras transgenic mice exhibit the following phenotype: 1) Newborn transgenic mice expressing v-ras^(Ha) (HK1 ras) exclusively in the epidermis show distinct wrinkled skin at 48 hours and are smaller than litter mates. 2) Juvenile HK1 ras transgenic mice exhibit progressive keratinization which peaks at 14 days. 3) The histotype of newborn HK1 ras mice reveals massive epidermal hyperplasia with up to 20-fold thickening of the epidermis. 4) By day 14 this progresses to massive hyperkeratosis. Both histotypes are pre-neoplastic, papillomatous, non-dysplastic and exhibit few appendages.

The HK1 ras transgenic mice develop benign tumors. Typical lesions appear within 10-12 weeks at single sites. The histotype of these tumors reveals a well differentiated squamous papilloma. Papillomas often appear at sites after wounding. Many of these papillomas are prone to regression. This regression phenomenon suggests that ras^(Ha) alone is insufficient to maintain even a benign phenotype and requires further events which may involve roles for additional oncogenes/antioncogenes. (Greenhalgh, et al., Induction of Epidermal Hyperplasia, Hyperkeratosis and Papillomas in Transgenic Mice by a Targeted v-Ha-ras Oncogene, Mol. Carcinog., Vol. 7, pp. 99-110 (1993)).

EXAMPLE 7 Targeting the fos Oncogene to the Epidermis of Transgenic Mice

Recent in vitro studies have shown that the v-fos gene can convert to malignancy primary keratinocytes or papilloma cell lines which expressed an activated ras^(Ha) (Greenhalgh, et al., PNAS, USA, Vol. 87, pp. 643-647 (1990); Greenhalgh and Yuspa, Mol. Carcinogen., Vol. 1, pp. 134-143 (1988)). This suggested that fos could play a later role in epidermal carcinogenesis and cooperate with the benign phenotype imparted by activated ras-^(Ha) expression. Although this alone was sufficient to initiate the establishment of HK1 fos transgenic mice with a view to mate with HK1 ras mice, two further studies have identified a role for fos in normal epidermal differentiation and thus highlights fos as an attractive target for perturbation. Using a c-fos/β-gal fusion gene Curran and co-workers (Smeyne, et al., Neuron, Vol. 8, pp. 13-23 (1992)) have shown significant fos expression in the differentiated layers of the epidermis and (Fisher, et al., Development, Vol. III, pp. 253-258 (1991)) have localized c-fos expression to a specific subset of granular cells.

Thus, fos may have an important role in the control of the final stages of keratinocyte differentiation. The putative perturbations of this normal role for c-fos in such specialized cells by v-fos can only be explored in the context of targeted expression in transgenic mice. In addition the c-fos proto-oncogene is known to function as a transcriptional regulator in conjunction with the c-jun/AP1 gene product and thus, while targeting ras^(Ha) represents studies of membrane signalling on neoplasia, targeting fos explores the role of transcriptional control on this process.

Thus, the fos protein coding sequence from the FBJ/FBR chimeric v-fos plasmid pFBRJ was inserted into the human keratin K1 targeting vector (FIG. 4). To discriminate expression of the v-fos transgene from that of the endogenous fos gene, a sequence encoding the human keratin K1 epitope (Sequence ID No. 5) was engineered onto the 5' end of the v-fos cassette.

HK1 fos transgenic mice exhibit the following phenotype: 1) A specific ear phenotype typically appears at 3-4 months initially in the wounded (tagged) ear and then becomes bilateral. 2) In several animals expressing severe phenotypes, the wounded ear lesion can grossly resemble a benign keratoacanthoma. 3) Alopecia and hyperkeratosis of the axilla often develop in older animals (approximately 1 year of age).

The histotypes of the HK1 fos mice are as follows: 1) The histotype of the initial ear lesions exhibits hyperplasia and hyperkeratosis, a pre-neoplastic pathology with few dysplastic cells and little evidence of further neoplastic progression. 2) At later stages the massive hyperkeratotic histotype resembles a benign keratoacanthoma.

Three HK1 fos transgenic mice lines have been established which develop an obvious pre-neoplastic ear phenotype at 3-4 months. The promotion stimulus derived from wounding (i.e., ear tag) appears to accelerate the appearance of this phenotype which eventually becomes bilateral. Also, it appears that friction in the axilla and inguinal area may also promote a pre-neoplastic hyperplastic/hyperkeratotic response after a significant latent period. Collectively these data support a fundamental role for the fos gene in normal keratinocyte differentiation and perturbation by v-fos results in pre-neoplastic differentiation disorders. In several HK1 fos mice severe ear lesions appear to progress to resemble benign keratoacanthomas. Although numbers are low at this time, that this is the resultant tumor type is consistent with a role for fos in the latter stages of terminal differentiation, and low numbers and latency suggest a requirement for additional events. (Greenhalgh, et ale, Hyperplasia, Hyperkeratosis and Benign Tumor Production in Transgenic Mice by a Targeted v-fos Oncogene Suggest a Role for fos in Epidermal Differentiation and Neoplasia, Oncogene, Vol. 8, pp. 2145-2157 (1993)).

EXAMPLE 8 Targeting HPV 18 E6 and E7 Gene Expression to the Epidermis

There is widespread evidence from clinical and epidemiological studies which implicate human papilloma viruses (HPV) in the etiology of certain squamous epithelial tumors in humans. HPV's have a specific tropism for squamous epithelial cells and different types of HPVs have specificity for the anatomic site that they infect. Additionally, within a specific subgroup of HPVs, certain types are associated with development of either benign (e.g., HPV6 and 11) or malignant (e.g., HPV-16 and 18) disease and this may center on the properties of the E6 and E7 genes. Through adaptation to the differentiation programs of the epithelia that they infect, HPVs have evolved a clever strategy for the production of infectious progeny. HPVs infect basal epithelial cells but do not undergo lytic replication in this compartment, thus, the germinative pool of cells is not subjected to the cytopathic effects of late viral gene expression. Production of virus only occurs in terminally differentiated cells that have lost proliferative potential and will be desquamated into the environment.

This strategy not only provides for the spread of mature viral particles, but ensures their continuous production by replenishment with cells from the basal compartment. Since the life cycle of the virus is so tightly linked to all stages of differentiation of squamous epithelial cells, establishment of successful culture systems has been difficult. To date, these host factors, coupled with regulatory mechanisms present within papilloma virus genomes themselves have also hindered attempts to observe pathological effects of HPV gene expression in squamous epithelia in transgenic mice. These restrictions on utilization of the transgenic mouse model have been overcome with the ability to specifically target HPV gene expression to squamous epithelia using the human keratin K1 targeting vector.

In the example provided, the sequence encoding the HPV-18 E6 and E7 ORF was inserted at the Bam HI and Cla I sites of the human keratin K1 targeting vector polylinker to create the HK1.E6/E7 transgene, by employing a 5' E6 specific oligo #1 (5'-CCCCGGGATCGATCTGGATCAGCCATTGTTGC-3') and a 3' E7 specific oligo #2 (5'-CCCGGGAGATCTCACAATACTATGGCGCGC-3'). The E6/E7 ORF was initially obtained by PCR amplification of HPV18 nco plasmid DNA using oligos #1 and #2, and the reaction conditions described previously, Kopan, et al., Retinoids as Important Regulators of Terminal Differentiation: Examining Keratin Expression in Individual Epidermal Cells at Various Stages of Keratinization, J. Cell Biol., Vol. 105, pp. 427-440 (1987); Hosomi, et al., Regulation of Terminal Differentiation of Cultured Mouse Epidermal Cells by 1a, 25-dihydroxyvitamin D3, Endocrinology, Vol. 113, 1950-1957 (1983). The PCR product was digested with Bgl II and ClaI, paper purified, subcloned into the human keratin K1 targeting vector and sequenced employing standard techniques (FIG. 5).

Generation and Identification of Transgenic Mice

The control of HPV gene expression is tightly linked to the program of differentiation in squamous epithelia and is species-restricted. In order to overcome these limitations, and generate an in vivo transgenic mouse model, an epidermal targeting vector was employed, based on HK1, to express the transforming genes E6 and E7 of HPV 18 exclusively in the epidermis.

Plasmid DNAs were prepared by standard techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory) (1982)). The HK1.E6/E7 transgene was released from the pGem3 plasmid by digestion with EcoRI and the 7 kb fragment isolated by agarose gel electrophoresis and paper purification. The purified HK1.E6/E7 DNA concentration was adjusted to 2 ng/μl in 10 mM Tris HCL, 0.25 mM EDTA pH 7.5 and subjected to 40,000 rpm ultracentrifugation for 1 hour to remove debris prior to microinjection. Mouse embryos were isolated as described previously (Hogan, et al., Manipulating the Mouse Embryo. A Laboratory Manual (Cold Spring Harbour, N.Y.: Cold Spring Harbour Laboratory) (1986)). DNA was microinjected into the pronuclei of one cell mouse embryos, obtained from 10-12 g female ICR mice mated to male FVB mice supplied by Sasco animal facility (Houston, Tex.). After microinjection, 15-20 surviving embryos were transferred to the oviduct of ICR pseudo pregnant foster mothers and normal gestation allowed. Transgenic mice were confirmed by tail DNA isolation (Hogan, et al., Manipulating the Mouse Embryo. A Laboratory Manual (Cold Spring Harbour, N.Y.: Cold Spring Harbour Laboratory) (1986)), and PCR analysis using vector-specific oligos #3 (5'-TGGTCCACTTGGGATTGGTG-3') and #4 (5'-GGAGTCTCATAGCCATGG-3') specific for the first intron of the expression vector.

Preparation and Analysis of RNA

Mouse epidermis, prepared from 1-5 day old neonates as described (Yuspa, et al., Epidermal Cell Culture, Transplantation Proc., Vol. 12, Suppl. 1, pp. 114-122 (1980), together with surgically removed lesions and organs, was routinely stored in liquid nitrogen prior to total RNA isolation employing the RNAzol (Biotex Labs, Houston) protocol. Total RNA was subjected initially to Northern analysis to determine the expression of the HK1.E6/E7 transgene.

Following formaldehyde agarose gel electrophoresis and transfer to nitrocellulose, filters were hybridized to a random-primed sequence specific for the 3' non-coding region of the vector (specific activity 2×10⁸ cpm), and washed to a final stringency of 0.5× standard sodium citrate/0.1% SDS at 68° C. prior to autoradiography at -70° C. using Kodak X-Omat AR5 film with intensifying screen. Total RNA was also analyzed for HK1.E6/E7 expression by reverse transcriptase PCR (RT/PCR) employing the BRL preamplification system (BRL Bethesda, Md.) and, following manufactures instructions, cDNA was generated using 5 μg of total RNA and 200 ng of a HK1 3' non-coding specific oligo (oligo #5 (5'-ATCGACCTCGGTCTTGCC-3')). This cDNA was amplified employing oligos #1 and #2 specific for E6 and E7 as described (Greenhalgh, et al., Targeted Expression of v-ras^(Ha) to the Epidermis of Transgenic Mice Induces Hyperplasia, Hyperkeratosis and Papillomas, Mol. Carcinog., Vol. 7, pp. 99-110 (1993)), using the absence of reverse transcriptase to account for DNA contamination, or using oligo #6 (5'-CCCGGCTTCGAATTTGCCTCCTTCATTC-3'), specific for vector sequences 5' to the intron.

This latter reaction allowed for both assessment of contaminating DNA and the cloning, into pGem3 at the Cla1 sites, of PCR amplified (oligos #6 and #2), Csp45/ClaI digested cDNA representing the predominant HK1.E6/E7 transcript which was then subsequently sequenced. To show that full length E6/E7 transcripts were produced, the HK1.E6/E7 cDNA was amplified using a 5' oligo #7 (5'-TATACCCCATGCTGCATGCC-3') that was specific for the deleted sequence produced by the alternative splicing that creates the E6*/E7 transcript, and oligo #2. To confirm that this transcript truly represented full length E6, this PCR product was digested with Xba I.

Microinjection of the HK1.E6/E7 construct gave numerous viable offspring and PCR analysis of tail tip DNA, employing vector-specific oligos #3 and #4, identified the presence of the transgene in seven founders. Northern analysis, using a vector specific probe, confirmed that three lines were high expressors. These lines were propagated to homozygosity. HK1.E6/E7 transgenic mice were indistinguishable from normal, non-transgenic siblings for the majority of their adult life, but once older than 1 year (and more typically 16-20 months) HK1.E6/E7 mice began to exhibit small lesions underlying the fur. These lesions were initially identified by a roughness and a subtle rigidity of the skin, which occurred predominantly on the nape of the neck, back and dorsal ear surface. Following careful shaving and use of a depilatory cream, HK1.E6/E7 mice exhibited up to 15-20 pinpoint, raised structures, often with a scaly, keratotic cap.

These lesions were prone to regression, some disappearing within 6-8 weeks of the initial observation. Also, unlike transgenic mice expressing v-ras^(Ha) or v-fos, the HK1.E6/E7 lesions did not appear to be associated with a wound promotion stimulus (e.g., ear tag). Not all animals were documented to possess these phenotypes. However due to the subtle nature of the lesions and their propensity to regress, these animals probably possessed unidentified lesions. Also, a second type of lesion appeared which was larger and more typical of a benign squamous cell papilloma. These papillomas appeared in old mice (18-21 months), grew slowly, and did not convert to malignant conversion.

Histopathology of HK1.E6/E7 Lesions and Keratin Immunofluorescence

Initially, the histotype of HK1.E6/E7 transgenic epidermis was indistinguishable from normal, non-transgenic siblings for all three lines, as was gingiva, vagina, anus and forestomach, which are possible alternate sites for mouse K1 expression. The earliest indications of HK1.E6/E7 induced pathology occurred in a 322 F₁ mouse #635 at approximately 10 months of age. The histotype is similar to a wart like lesion typically induced by HPV, consisting of a marked hyperplasia and hyperkeratosis, with a prominent stratum granulosum, and having distinct verrucous appendages descending from a highly keratotic plug. All histotypes of these subtle, verrucous lesions were identical.

Conversely, the second lesion histotype was confirmed as a typical, squamous cell papilloma with no areas of malignant conversion or carcinoma in situ. Frozen sections from epidermis, typical warts and papillomas were assessed for mouse keratin K1, K6, K13 and K14 expression by double label immunofluorescence. HK1.E6/E7 epidermis expressed keratins in an identical manner to normal, nontransgenic epidermis. While novel expression of the hyperproliferative keratin MK6 was observed in all lesions, they also retained MK1 expression, loss of which is observed in malignant conversion, indicating the benign nature of these lesions. Also, these lesions did not express K13, an early marker for malignant conversion. Thus both histotype and specific keratin markers confirmed the hyperproliferative but benign nature of the HK1.E6/E7 induced phenotypes.

Thus, it appears that the HK1.E6/E7 transgenic mice mimic not only the pathology, but also the epidemiology of HPV-16 or HPV-18 induced human disease, namely a long latency period between HPV induced preneoplastic disease and the onset of overt neoplasia.

The apparent delay and low phenotype frequency exhibited by these mice provides a relevant background to study the consequences of HPV expression during epithelial differentiation. In addition, these mice can be useful in assessing the efficacy of novel antisense pharmaceuticals which have been designed to inhibit expression of the E6 and E7 genes of HPV 18.

EXAMPLE 9 Analysis of HK1.E6/E7 Transcripts and ras^(Ha) Activation

Due to latency and low lesion frequency, coupled to the alternative splicing available to the HPV18 E6/E7 region analysis of the transcripts produced by HK1.E6/E7 transgene expression was carried out. cDNA was generated from newborn epidermis representative of each line or a squamous papilloma using oligo #5 and amplified using oligos #6 and #2 as defined in Example 8. The ability to amplify across an intron using oligo #6 allowed assessment of DNA in RNA samples, but gave a slightly higher band size than that of the HPV.18 nco plasmid positive control, amplified by E6 and E7 specific oligos #1 and #2 as defined in Example 8. These experiments demonstrated the existence of two transcripts in epidermal and tumor RNA, with a similar upper to lower band intensity for epidermis and tumor. Subsequent Southern analysis showed that both of these PCR products were detected by an E6/E7 specific probe. That the more predominant lower 683 bp band represented the alternative splice E6*/E7 transcript was confirmed by cloning this amplified PCR product into a pGem3 vector and subsequent sequencing. The upper 873 bp band was confirmed to be a full length E6/E7 transcript by replacing oligo #6 with oligo #7 in the PCR amplification reaction. Oligo #7 is specific for the sequences lost on creation of E6*. This generated a 680 bp band in epidermal and papilloma RNA identical to the plasmid positive control. Diagnostic Xba I digestion of these PCR products give the expected 641 bp band, indicative of a full length E6 transcript. The verrucous lesions also possessed ratios of E6 to E6* transcripts identical to that of the ras^(Ha) papilloma.

Since the latency of lesion appearance suggested the requirement for events additional to the transgene expression, the ras^(Ha) oncogene was investigated for characteristic activating mutations. These have been well documented to occur spontaneously in mouse skin carcinogenesis and occur frequently in chemical carcinogenesis. cDNA generated from several verrucous lesions and squamous papillomas by random priming was amplified by c-ras^(Ha) specific oligos and the PCR product subjected to single-strand sequencing. All verrucous lesions analyzed had a normal sequence at codons 12, 13 and 61. However while possessing a normal codon 12 and 13, the anogenital papilloma possessed an A→T transversion at codon 61. Conversely the neck papilloma had normal codons 12 and 61 but possessed a G→T mutation within codon 13.

Previously, it was observed that a HK1.ras transgene has profound effects in newborn but not adult epidermis (Greenhalgh, et al., Targeted Expression of v-ras^(Ha) to the Epidermis of Transgenic Mice Induces Hyperplasia, Hyperkeratosis and Papillomas, Mol. Carcinog., Vol. 7, pp. 99-110 (1993)). That data indicated the necessity for cooperating genetic events additional to HK1.E6/E7 expression that lead to overt lesion appearance and eventual neoplasia. It is of interest that while the host factors that cooperate with HK1.E6/E7 to produce the verrucous lesions remain unknown, in the progression to a benign squamous cell papilloma, the ras^(Ha) oncogene was found to be activated by an A→T transversion at codon 61 (c-ras^(Ha61)). This is typical of the mutations found in spontaneous or chemical activation of the murine c-ras^(Ha) protooncogene. A second activating mutation (G→T) was detected at codon 13 (c-ras^(Ha13)) in a histologically identical squamous cell papilloma. These mutations were not detectable in the verrucous lesions and this suggests that squamous papillomas develop from a cooperation between the HK1.E6/E7 and the spontaneously activated endogenous c-ras^(Ha) oncogene. Cooperation between HPV-16 and 18 and ras^(Ha) has been observed previously in vitro and centered on E7 expression (Woodworth, et al., Recombinant Retrovirus Encoding Human Papillomavirus Type 18 E6 and E7 Simulate Proliferation and Delay Differentiation of Human Keratinocytes Early After Infection, Oncogene, Vol. 7, pp. 619-626 (1992)).

This is of note since, HK1.E6/E7 expression in the transgenic mouse epidermis is predominantly E6*/E7. This bears significance to the relevance of HK1.E6/E7 mice as a model for the human condition as approximately 10% of cervical carcinomas contain ras^(Ha) mutations. Moreover, HPV-16 has recently been associated with squamous cell carcinomas etiology on sun exposed sites (Pierceall, et al., Presence of Human Papillomavirus Type 16 DNA Sequences in Human Non-melanoma Skin Cancers, J. Invest. Dermatol., Vol. 97, pp. 880-884 (1991)), up to 46% of which contain UV-induced activating ras^(Ha) mutations (Pierceall, et al., Ras Gene Mutations and Amplification in Human Nonmelanoma Skin Cancers, Mol. Carcinog., Vol. 4, pp. 196-202 (1991)). Also, as HPV represents a serious problem for immune suppressed individuals (Broker, et al., A Molecular Portrait of Human Papillomavirus Carcinogenesis, pp. 197-207, In: Molecular Diagnostics of Human Cancer, Cancer Cells 7, Cold Spring Harbor, N.Y, (1989)), and given the frequency of ras^(Ha) activation in human cancers (Bos, The ras Gene Family and Human Carcinogenesis, Mutant Res., Vol. 198, pp. 255-271 (1988)), the mating of HK1.E6/E7 mice with transgenic mice expressing activated ras^(Ha) (HK1.ras) could prove a powerful system to explore gene synergism in vivo.

The HK1.E6/E7 transgene, being derived from HPV-18 DNA, contains an alternative splice site which augments the expression of E7 at the expense of a full length E6 transcript, producing the non-transforming E6* (Sedman, et al., The Full Length E6 Protein of Human Papillomavirus Type 16 has Transforming and Transactivating Activities and Cooperates with E7 to Immortalize Keratinocytes in Culture, J. Virol., Vol. 65, pp. 4860-4866 (1991)). Careful analysis of the HK1.E6/E7 transcripts showed that for all three lines, epidermis, verrucous lesions and papillomas expressed similar and predominant levels of the E6*/E7 transcript. The low levels of the full length E6 transcript, barely detectable in newborn epidermal RNA, remained unchanged in lesions or papillomas on comparison to the E6*/E7 transcript. Thus, the transgenic mice may be considered predominantly E7, hence the potential for cooperation with c-ras.

Since the data suggests the necessity of secondary and tertiary events in the low frequency and long latency for the development of HPV associated pathology in our mice may be a consequence of low level full length E6 expression. The full length E6, in addition to cooperating with E7 in immortalizing human keratinocytes, is capable of transforming murine NIH 3T3 cells in vitro (Sedman, et al., The Full Length E6 Protein of Human Papillomavirus Type 16 has Transforming and Transactivating Activities and Cooperates with E7 to Immortalize Keratinocytes in Culture, J. Virol., Vol. 65, pp. 4860-4866 (1991)), and the BPV E6 can immortalize human epithelial cells alone. Moreover, the HPV16 E6 gene was shown to cooperate with ras^(Ha) and immortalize keratinocytes apparently independent of E7 (Story and Banks, Human Papilloma Virus Type 16 E6 Cooperates with EJ-ras to Immortalize Primary Mouse Cells, Oncogene, Vol. 8, pp. 919-924 (1993)). Therefore, the combination of functional E6 and E7 together with ras^(Ha61), may be required to achieve either higher lesion frequencies (both verrucae and papillomas), and malignant conversion in this transgenic epidermal model.

Accordingly, it will be useful to produce an HK1.E6 mouse for mating purposes with the HK1.E6/E7 (i.e., E6*/E7) mice and HK1.ras. Furthermore, if progression to malignancy in HPV-16 and 18 infected tumors is contingent on E6 and E7 binding to the p53 and Rb tumor suppressor proteins, thereby inactivating their function), the mating of HK1.E6/E7 mice (i.e., E6*/E7) to those null for the p53 gene would give a functional in vivo murine equivalent to the putative E6/p53 interaction found in humans.

EXAMPLE 10 Production of Transgenic Mice Expressing TGF-α in the Epidermis

Transforming growth factor alpha (TGF-α) is a cytokine with structural and functional characteristics similar to epidermal growth factor (EGF). Both TGF-α and EGF bind to the epidermal growth factor receptor (EGF-R) and stimulate the tyrosine kinase cascade. TGF-α is expressed by both normal and transformed cells and causes proliferation of cultured keratinocytes. In vivo, TGF-α induces angiogenesis and is more potent than EGF in accelerating wound healing. In normal human skin, expression of TGF-α occurs in all layers of the epidermis and in certain areas of the appendages. Several cutaneous diseases such as psoriasis, squamous cell carcinoma, and congenital bullous ichthyosiform erythroderma have been associated with altered expression of TGF-α.

To determine whether altered expression of TGF-α plays a role in the pathogenesis of these diseases, the protein coding sequence of human TGF-α was inserted into the human keratin K1 targeting vector (FIG. 6).

Construction of the HK1.TGF-α Transgene

The targeting vector (pHK1) is derived from a 12 kb EcoRI fragment of the HK1 gene from lambda clone c55 (Johnson, et al., Structure of a Gene for the Human Epidermal 67 kDa Keratin, Proc. Natl. Acad. Sci. USA, Vol. 82, pp. 1896-1906 (1985)), that has been found to direct expression in a tissue-specific and developmental-specific fashion (Greenhalgh, et al., Induction of Epidermal Hyperplasia, Hyperkeratosis and Papillomas in Transgenic Mice by a Targeted v-Ha-ras Oncogene, Mol. Carcinog., Vol. 7, pp. 99-110 (1993); Greenhalgh, et al., Hyperplasia, Hyperkeratosis and Benign Tumor Production in Transgenic Mice by a Targeted v-fos Oncogene Suggest a Role for fos in Epidermal Differentiation and Neoplasia, Oncogene, Vol. 8, pp. 2145-2157 (1993)).

As previously described, this 10.8 kb fragment is missing certain regulatory elements which restrict expression to the differentiated compartment of the epidermis. This truncation allows expression of the transgene to be directed to 20-30% of the proliferative compartment of the epidermis (Rosenthal, et al., A Human Epidermal Differentiation Specific Keratin Gene is Regulated by Calcium but not Negative Modulators of Differentiation in Transgenic Mouse Keratinocytes, Cell Growth and Diff., Vol. 2, pp. 107-113 (1991)).

Briefly, through the use of PCR, all HK1 exons and introns, with the exception of the first intron, have been deleted and a polylinker with unique restriction enzyme sites was introduced 3' of the first intron. Thus, the targeting vector retains all of the 5' flanking sequence up to but not including the ATG, the first intron including the intron/exon splice site, and the 3' non-coding and flanking sequences after the TAA codon (FIG. 6). The EcoRI sites at the vector ends were retained and the vector cloned into pGem3 for bacterial amplification and replication. The sequence encoding human TGF-α cDNA (plasmid phTGF1-10-925 kindly provided by Dr. Graeme Bell, University of Chicago) was introduced into the HK1 vector by engineering unique Bam HI and Cla I restriction enzyme sites onto the 5' and 3' ends of the TGF-α cDNA by PCR through the use of TGF-α specific oligonucleotides #1 (5'-AAACGCGGATCCATGGTCCCCTCGGCTGGA-3') and #2 (5'-CCATCGATGGTCAGACCACTGTTTCTGA-3'). This product was amplified and subcloned into the TA cloning vector (Invitrogen). The 50 μl reaction consisted of 5 μl of 10× PCR buffer (Invitrogen), 1 μl of 25 mM dNTP's, 1 μl of each primer and 4 units of Thermus aquaticus (Taq) DNA polymerase (Cetus). 30 PCR cycles of the following profile were used: 1 minute at 94° C., 2 minutes at 55° C., 3 minutes at 72° C., and finally 7 minutes at 74° C. After confirmation of the correct sequence, this 500 bp Bam HI-Cla I fragment of the TGF-α cDNA was subsequently subcloned into the Bam HI and Cla I sites of the HK1 targeting vector (FIG. 6).

Generation and Identification of Transgenic Mice

The pHK1-TGF-α plasmid was digested with EcoRI in order to release the transgene and the 6.7 kb fragment was isolated by agarose gel electrophoresis and paper purified. HK1.TGF-α DNA concentration was adjusted to 2 ng/ml in 10 mM Tris HC1, 0.25 mM EDTA pH 7.5 and ultracentrifuged at 40,000 rpm for 30 minutes. One cell mouse embryos were isolated from 10-12 gram ICR female mice (Charles River) mated to FVB male mice (Frederick Cancer Research Facility) and the transgene DNA was microinjected into the male pronucleus of the embryos (Johnson, et al., The Structure of the Gene for the Human Epidermal Keratin of 67,000 Molecular Weight, Proc. Natl. Acad. Sci. USA, Vol. 82, pp. 1896-1900 (1985)). These embryos were implanted into ICR pseudopregnant foster mothers. PCR analysis using oligonucleotides #3 (5'-TGGTCCACTTGGGATTGGTG-3') and #4 (5'-GGAGTCTCATAGCCATGG-3') specific for the first intron of the expression vector (FIG. 6) were used to confirm the presence of the transgene in tail tip DNA from 21 day old founder mice. The 100 μl PCR reaction consisted of 10 μl 10× buffer (Cetus), 2 μl each 10 mM dNTP, 1 μl diluted DNA, 1 μl each of 5' and 3' primers at 200 ng/μl, 78 μl H₂ O, and 1 μl Taq DNA polymerase (Cetus). Samples were heated to 95° C. for 5 minutes, then 30 cycles of amplification consisted of denaturation at 95° C. for 30 seconds, annealing at 60° C. for 1 minute and extension at 72° C. for 1 minute, the final extension was at 72° C. for 15 minutes.

Northern and Reverse Transcriptase/PCR Analysis (RT/PCR)

Skins from newborn and adult mice (transgenic and control) were floated on 0.25% trypsin at 4° C. for 18 hours, after which the epidermis and dermis were separated as independent sheets (Yuspa, et al., Epidermal Cell Culture, Transplantation Proceedings, Vol. 12, Suppl. 1, pp. 114-122 (1980)). Total RNA was isolated from the epidermis using the RNAzol protocol (Biotex Laboratories). Following formaldehyde agarose gel electrophoresis and transfer to nitrocellulose, filters were hybridized with ³² P-labelled random-primed probes corresponding to the 3' non-coding (3' NC) human keratin 1 sequence, or a TGF-α specific sequence. Filters were washed to a stringency of 0.5 SSC 0.1% SDS at 68° C. prior to autoradiography at -70° C. using Kodak X-O-Mat AR5 film with an intensifying screen. To account for loading discrepancies, filters were reprobed with a probe specific for βactin.

Total RNA was also isolated from papillomas and stomach, brain, liver, kidney, and spleen and assessed for HK1.TGF-α expression by RT/PCR analysis. cDNA was generated by incubating 2-μg of total RNA with reverse transcriptase (Life Sciences, St. Petersburg, Fla.) according to the manufacturer's instructions and 200 ng of 3' NC specific oligo #5 (5'-ATCGACCTCGGTCTTGCC-3') at 37° C. for 2 hours. The resultant cDNA was amplified by PCR using TGF-α specific oligos #1 and #2 as outlined above to give an approximate 500 bp band indicative of HK1.TGF-α expression. Control reactions without reverse transcriptase were included to account for DNA contamination of RNA samples.

Radioimmunoassay

Epidermis from transgenic and control newborn mice was separated from dermis by heating in 0.15M NaCl at 68° C. for 2 minutes. Protein was extracted using three different methods. Acid/ethanol extraction as described by Robert, et al., Transforming Growth Factors: Isolation of Polypeptides from Virally and Chemically Transformed Cells by Acid/ethanol Extraction, Proc. Natl. Acad. Sci., USA, Vol. 77, pp. 3494-3498 (1980)), cytosol/membrane extraction as described by Imamato, et al., Evidence for Autocrine/paracrine Growth Stimulation by Transforming Growth Factor-α During the Process of Skin Tumor Promotion, Mol. Carcinog., Vol. 4, pp. 52-60 (1991), and total epidermal protein extraction.

Total epidermal protein extracts were prepared by homogenization in 0.0625M Tris-HC1, pH 6.8, 5% SDS, 10% glycerol, and 20% 2-mercaptoethanol followed by heating at 95° C. for 10 minutes. After a brief centrifugation step to remove insoluble material, the supernatant was dialyzed against TBS buffer containing 1 mM EDTA and 0.1% Triton X-100. Quantification of protein was performed as described by the Bradford assay and quantified by a TGF-α radioimmunoassay (Biomedical Technologies, Inc., Stroughton, Mass.).

Immunoprecipitation

Newborn HK1.TGF-α or control skin samples were trypsinized overnight at 4° C. and once separated from dermis (Yuspa, et al., Epidermal Cell Culture, Transplantation Proceedings, Vol. 12, Suppl. 1, pp. 114-122 (1980)), the epidermis was incubated in low calcium, cysteine-free EMEM containing 1 mCi S³⁵ -cysteine overnight at 37° C. The epidermis was then washed three times with PBS, centrifuged, and the pellet lysed in Tris-SDS-glycerol-mercaptoethanol buffer, followed by heating to 95° C. for 10 minutes and centrifugation. The resultant supernatant was dialyzed with RIPA buffer (1% NP-40, 0.1% aprotinin, 1% deoxycholate, 0.1% SDS, 150 mM sodium chloride, 1 mM EDTA, 1 mM PMSF, 20 mM MOPS pH 7.6) at room temperature for 24 hours.

Samples with 3×10⁶ cpm were adjusted to a volume of 1 ml with RIPA buffer and normal rabbit serum (20 μl) added; the samples were incubated for 1 hour at 4° C. Continuing this pre-clearing step, 50 μl of goat antimouse IgG-agarose was then added for 1 hour at 4° C. After centrifugation, the supernatant was collected and incubated with 2 μg of TGF-α antibody (Oncogene Science) for 3 hours at 4° C. followed by addition of 50 μl of goat anti-mouse IgG-agarose and incubation for a further hour at 4° C. After centrifugation, the pellet was washed repeatedly with RIPA buffer and buffer containing 2.5M KCl and recentrifugated. Samples were resuspended in solubilization buffer and 50 μl aliquots were incubated with 0.32M iodoacetamide for 15 minutes at 37° C. Samples were then run on 12.5% tricine-SDS-PAGE gels and fixed in 40% methanol/10% acetic acid. After incubation with Enhance (Dupont) followed by water (30 minutes each), gels were dried and fluorographed.

Tissue Histology

Skin, liver, mammary gland, tongue, stomach, vagina, gingiva, and pancreatic tissues were fixed in Carnoy's solution (chloroform/acetic acid/ethanol 3:1:6 v/v) at 4° C. overnight, transferred to 95% ethanol, embedded in paraffin, sectioned and stained with hematoxylin and eosin.

Immunofluorescence

Biopsy samples were frozen in OCT at -70, sectioned at 4-6 microns and mounted onto glass slides. Slides were washed twice in PBS for 10 minutes and rinsed in distilled water for 2 minutes. Monospecific antikeratin antibodies from two species (Roop, et al., Regulated Expression of Differentiation Associated Keratins in Cultured Epidermal Cells Detected by Monospecific Antibodies to Unique Peptides of Mouse Epidermal Keratins, Differentiation, Vol. 35, pp. 143-150 (1987); Roop, et al., Transcriptional Control of High Molecular Weight Keratin Gene Expression in Multistage Mouse Skin Carcinogenesis, Cancer Res., Vol. 48, pp. 3245-3252 (1988); Nischt, et al., Abberant Expression During Two Stage Mouse Skin Carcinogenesis of a Type I 147-kDa Keratin, K13, Normally Associated with Terminal Differentiation of Internal Stratified Epithelia, Mol. Carcinogen., Vol. 1, pp. 96-108 (1988); Smith, et al., Differential Keratin Gene Expression in Developing, Differentiating, Preneoplastic and Neoplastic Mouse Mammary Epithelium, Cell Growth and Diff., Vol. 1, pp. 161-170 (1990)), were applied overnight at room temperature.

The following dilutions were prepared in 12% BSA/PBS (v/v): guinea pig anti K14 1:2000, rabbit anti K1 1:500, rabbit anti K6 1:2000, rabbit anti K13 1:500, rabbit anti loricrin (Mehrel, et al., Identification of a Major Keratinocyte Cell Envelope Protein, Loricrin, Cell, Vol. 61, pp. 1103-1112 (1990)), 1:500 and rabbit anti filaggrin (Rothnagel, et al., The Structure and Expression of the Mouse Epidermal Filaggrin Precursor Gene, J. Biol. Chem., Vol. 262, pp. 15643-15648 (1987)) 1:500. Sections were washed as above and secondary biotinylated goat anti guinea pig 1:100 (Vector), along with normal goat 1:100 and swine serum 1:100, were applied for 30 minutes at room temperature Sections were washed again and 1:400 Streptavidin-Texas Red (Gibco) and 1:40 swine anti rabbit FITC (Dakopatts) were applied for 30 minutes at room temperature. Sections were washed, air dried, and cover-slipped with Fluoromount (Fisher Biotech).

Bromodeoxyuridine Uptake and Staining

Samples were processed as previously described (Huitfeldt, et al., Altered Regulation of Growth Expression of Differentiation Associated Keratins in Benign Mouse Skin Tumors, Carcinogens, Vol. 12, pp. 2063-2067 (1991)). Briefly, newborn transgenic and control mice were injected (i.p.) with BrdUrd (Sigma) 250 mg/kg in 0.9% NaCl and sacrificed after 2 hours. Dorsal and ventral skin was fixed in 70% ethanol at 4° C. and embedded in paraffin. Sections were cut, deparaffinized, and soaked in 2N HCl for 30 minutes. Sections were then washed in PBS, placed in 70% ethanol with 0.1M Tris twice for 10 minutes, switched to 70% ethanol twice for 5 minutes, rehydrated, and dried. Sections were incubated with undiluted FITC-conjugated monoclonal antibody to BrdUrd (Becton-Dickinson) mixed with guinea-pig antiserum to K14 (diluted 1:2000) for 20 hours. Sections were washed twice in PBS for 10 minutes, rinsed in distilled water, incubated with 1:100 goat anti guinea pig biotinylated antibody (Vector) for 30 minutes. After washing, 1:400 Streptavidin-Texas Red (Gibco) was applied for 30 minutes and sections were again washed, dried, and mounted with Fluoromount (Fisher Biotech).

Phenotypic Expression

Injection of the HK1.TGF-α construct into embryos resulted in phenotypic founders that were quite similar to that of ras^(Ha). PCR analysis of tail DNA using oligonucleotides specific for the first intron of HK1 (oligo #3 and #4,) confirmed the presence of the transgene in 10 founders. Subsequent northern analysis of F1 neonatal epidermal RNA confirmed that eight were expressors (1960, 1970, 1981, 2032, 2034, 2106, 2113, 5418). Seven of the founders and their progeny had a markedly thickened, opalescent skin at birth, and this phenotype persisted until growth of the first coat of hair. Founder 1981 had a very subtle phenotypic pattern that became more apparent in the F₁ generation. Affected newborn pups were often smaller in size compared to normal littermates and at 6-8 days of age, the HK1.TGF-α pups exhibited precocious eye opening compared to 10-12 days in normal litter mates. By 7-8 days of age, control littermates had growth of the first coat of hair whereas transgenic pups had diffuse alopecia with thickening and scaling of the skin that persisted until days 16-21. Five lines (1970, 2032, 2034, 2113 and 5418) had fine, sparse hair as compared to normal.

Three lines (1970, 2032, and 2113) retained the phenotype of thick, scaly skin as adults. The adult phenotype varied from uniform involvement of the abdominal and genital areas to focal involvement of areas like the head and neck, and may have been induced and sustained by trauma or mechanical irritation. These lines also demonstrated areas of hyperkeratosis and the appearance of spontaneous papillomas at 2-5 months of age. Generally, papillomas appeared at sites prone to wounding (ear tag) or repeated trauma (scratching and biting).

The founder mouse 2032 exhibited small papillomas in the inguinal region, with spontaneous resolution in 2-3 weeks. A typical derivative from the 1970 line had the appearance of multiple small papillomas on the upper back and ears that became large, ulcerative papillomas over a 5 month period, but then began to regress. This regression of papillomas was frequently observed. Autopsies of mice from several different lines demonstrated no gross changes in the mammary gland, liver, pancreas, oral epithelia, or forestomach and the HK1.TGF-α mice appeared to have a normal lifespan.

Histology

Histologic examination of newborn, 8 day old, and adult phenotypic transgenic skin showed marked epidermal hyperplasia with hyperkeratosis of the stratum corneum, preservation of the granular layer, and hypertrophy of the stratum malpighii, and relatively fewer hair follicles as compared to normal. The dermis was somewhat thinner than normal. No abnormal histologic changes were noted in the mammary gland, liver, pancreas, forestomach, or oral mucosa. Biopsies of the papillomas showed marked hyperkeratosis, acanthosis, papillomatosis, and a moderate dermal inflammatory infiltrate, consistent with a well differentiated squamous papilloma. Minimal dysplasia was occasionally seen focally but no frank carcinoma in situ was present.

Expression of HK1.TGF-α in Epidermis and Papillomas

Expression of HK1.TGF-α in the epidermis of phenotypic newborn pups and adults was confirmed by northern analysis of F1 neonatal RNA using a vector specific 3' non-coding (3' NC) probe. A 0.9 kb band, corresponding to the expected size of the HK1.TGF-α mRNA, was seen in all phenotypic epidermis. Identical results were obtained with a TGF-α specific probe. After reprobing with a βactin probe to assess for loading discrepancies, the more severely affected lines demonstrated greater intensity of signals. RT/PCR analysis was employed to detect HK1.TGF-α transcripts in phenotypic adult epidermis and papillomas, and identified the expected 500 bp band. This technique was primarily employed to assess other tissues for transgene expression and this band was absent in experiments involving template RNA isolated from stomach, brain, kidney, liver, lung and spleen, consistent with the expression properties of the vector and the absence of pathology outside the epidermis.

To assess HK1.TGF-α protein expression, ³⁵ S labelled epidermal extracts from phenotypic and control mice were analyzed by immunoprecipitation. In the control sample, a faint 21 kD band that corresponded to the relative mobility of the precursor endogenous TGF-α species was seen. In contrast, transgenic samples had a prominent band appearing around 15 kD which corresponded to the relative mobility of a TGF-α degradation product remaining after cleavage of mature 6 kD TGF-α species. To quantitate the level of human TGF-α, radioimmunoassays were performed. The concentration of human TGF-α in transgenic epidermal extracts varied between 2.4˜16.0 ng/mg protein, and the severity of the skin changes correlated with the level of TGF-α expression. Using acid/ethanol extraction, human TGF-α protein levels were enriched approximately 10-fold in the transgenic epidermis. Fractionation into epidermal cystolic and membrane components showed demonstrable increases in both compartments with the majority of the protein appearing in the cystolic fraction.

HK1.TGF-α Transgenic Mice Demonstrate Aberrant Keratin Expression and Hyperproliferation of the Epidermis.

To assess potential changes in markers of epidermal differentiation and proliferation, frozen sections from normal and transgenic mouse skin and papillomas were analyzed by double-label immunofluorescence (IF) with antibodies to K1, K6, K13, K14, loricrin and filaggrin. Under normal conditions, K14 is expressed in basal cells and is transcriptionally down-regulated once cells commit to terminal differentiation (Mehrel, et al., Identification of a Major Keratinocyte Cell Envelope Protein, Loricrin, Cell, Vol. 61, pp. 1103-1112 (1990)). However, K14 staining is seen to persist into upper layers of the epidermis because of the inherent stability of keratin proteins. K1, an early marker of terminal differentiation, is detected throughout the suprabasal layers, as well as in some basal cells (Roop, et al., Transcriptional Control of High Molecular Weight Keratin Gene Expression in Multistage Mouse Skin Carcinogenesis, Cancer Res., Vol. 48, pp. 3245-3252 (1988)), however, expression in basal cells occurs post-mitotically (Huitteldt, et al., Altered Regulation of Growth Expression of Differentiation Associated Keratins in Benign Mouse Skin Tumors, Carcinogen., Vol. 12, pp. 2063-2067 (1991)).

While normal control and transgenic epidermis had the appropriate expression of K14, expression of K1 was delayed in transgenic epidermis and papillomas, presumably due to expansion of the proliferative compartment. K6, a marker of hyperproliferation (Weiss, et al., Monoclonal Antibody Analysis of Keratin Expression in Epidermal Diseases: A 48- and 56-Kilodalton Keratin as Molecular Markers for Hyperproliferative Keratinocytes, J. Cell Biol., Vol. 98, pp. 1397-1406 (1984)), and other pathological conditions (Sellheyer, et al., Inhibition of Skin Development by Over-expression of Transforming Growth Factor-β1 in the Epidermis of Transgenic Mice, Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 5237-5241 (1993)), was expressed throughout the epidermis of hyperplastic neonatal skin and in papillomas, but was restricted to expression in hair follicles in normal controls. Expression of both loricrin and filaggrin, late markers of terminal differentiation was not altered in hyperplastic skin. No expression of K13, an early marker of malignant conversion, was seen in papillomas, which also retained K1 expression confirming their benign nature.

To assess potential changes in the proliferative compartment of phenotypic epidermis, normal and transgenic mice were labeled with BrdUrd for 2 hours prior to sacrifice. Staining with an anti-BrdUrd antibody shows a marked increase in mitotically active cells in transgenic epidermis over controls. Control epidermis had 32.2 (±5.8) labeled nuclei per mm, whereas transgenic epidermis had 60.5 (±10.6) labeled nuclei per mm. In addition, labeled nuclei were detected in suprabasal epidermal cells of HK1.TGF-α mice, confirming expansion of the proliferative compartment into the stratum spinosum.

In summary, the expression of human TGF-α was successfully targeted exclusively to the epidermis of transgenic mice using a vector derived from the HK1 gene. Eight transgenic founder lines were established which exhibited similar characteristics, with variations in severity dependent upon the expression levels of TGF-α. Over-expression of TGF-α in the epidermis resulted in transgenic mice whose phenotypes were immediately obvious at birth. These affected mice were smaller and had a markedly thickened and wrinkled skin, sparse thin hair, and epidermal hyperplasia and hyperkeratosis.

This example documents the potential pathological consequences of deregulated expression of TGF-α in the epidermis. Therapeutic strategies designed to inhibit TGF-α expression or interfere with its signal transduction pathway would be expected to prove efficacious in the treatment of certain hyperproliferative skin diseases, and this transgenic model would be expected to be useful in assessing these protocols.

EXAMPLE 11 Effect of on EGFR Expression in Transgenic Mice Expressing TGF-α in the Epidermis EGFR Levels and EGF Binding

In vitro studies suggest that TGF-α may regulate EGFR expression (Clark, et al., Epidermal Growth Factor Regulates the Expression of its Own Receptor, Proc. Natl. Acad. Sci. USA, Vol. 82, pp. 8374-8378 (1985); Earp, et al., Epidermal Growth Factor (EGF) Stimulates EGF Receptor Synthesis, J. Biol. Chem., Vol. 261, pp. 4777-4780 (1986); Kudlow, et al., Epidermal Growth Factor Stimulates the Synthesis of its Own Receptor in a Human Breast Cancer Cell Line, J. Biol. Chem., Vol. 261, pp. 4134-4138 (1986)), and in hyperproliferative conditions, is associated with persistent EGFR expression into the upper layers of the epidermis. Therefore, the distribution of EGFR in HK1.TGF-α transgenic mice was analyzed for potential alterations. Skin samples removed from normal adult mice showed intense immunoreactivity for EGFR in interfollicular epidermis, follicular epithelia and sebaceous glands. In adult HK1.TGF-α mice, the distant non-phenotypic skin had an immunolocalization pattern for EGFR identical to that observed in normal mouse skin. When phenotypic skin from three different HK1.TGF-α adult mice were evaluated for the presence of EGFR, a drastically different staining pattern was observed. Within these lesions no specific immunoreactivity for EGFR was detectable in either the epidermal layers at the surface or in the ridges extending into the underlying dermal regions. The validity of this observed negative immunostaining pattern was tested by including serial sections of these samples in three separate immunostaining experiments with sections that stained positive for EGFR. In addition, a longer reaction time with DAB only produced weak non-specific staining of adjacent dermis and the cornified layers. Nevertheless, relatively non-affected epidermis immediately adjacent to the phenotypic site showed the same strong immunostaining for EGFR as observed in both the normal adult epidermis and non-phenotypic epidermis at a more distal location.

When normal neonatal mouse skin was stained for EGFR, intense immunostaining was present in the interfollicular epidermis but only a faint immunostaining for EGFR was present in the growing hair follicles. By contrast, the hypertrophic sites of neonatal skin removed from a HK1.TGF-α mouse showed the complete absence of immunoreactive EGFR in the epidermis.

Additional data indicating that EGFR were downregulated in the phenotypic epidermis of newborn HK1.TGF-α mice were obtained from ¹²⁵ I EGF binding studies. Control newborn mice exhibited prominent ¹²⁵ I EGF binding in the basal and spinous layers of the epidermis as well as in hair follicles. A marked reduction in ¹²⁵ I EGF binding was observed in phenotypic skin. The highest levels of binding were detected in hair follicles and the adjacent epidermis.

EGFR Immunohistochemical Localization

For EGFR immunohistochemical staining, frozen sections were fixed in acetone for 10 minutes, rinsed in PBS and blocked in 5% goat serum for 20 minutes. Sections were then incubated for 18 hours at 4° C. in either EGFR polyclonal antisera or non-specific sera at a dilution of 1:500 using slight modifications to previously published protocols (Nanney, et al., Epidermal Growth Factor Receptors in Idiopathic and Virally Induced Skin Diseases, Am. J. Path., Vol. 140, pp. 915-925 (1992)). Skin sections were rinsed and reacted with the immune reagents in an avidin-biotin complex kit (Vector Laboratories). Immunoreactive sites in the mouse tissues were visualized using 3,3-diaminobenzidine as the chromagen.

EGFR Binding

Localization of EGFR binding by autoradiography was performed as previously described (Nanney, et al., Comparison of Epidermal Growth Factor Binding and Receptor Distribution in Normal Human Epidermis and Epidermal Appendages, J. Invest. Dermatol., Vol. 83, pp. 385-393 (1984)).

Approximately 1 mm² skin samples from newborn transgenic and control mice were incubated in 0.5 ml of Hank's balanced salt solution with 1 mg/ml BSA, 20 mM HEPES pH 7.4, and 10 ng/ml ¹²⁵ I EGF for 90 minutes at room temperature. Samples were then repeatedly washed in salt solution for 90 minutes at room temperature, fixed in 10% buffered formalin for 4 hours, and processed for light microscopy. Sections were placed in a 1:1 dilution of NTB-2 emulsion in water and exposed for 4 weeks at 4° C. The emulsion was developed using 1:1 dilution of Kodak Dektol for 2 minutes at 15° C., water stop bath for 20 seconds at 15° C., and 1:3 dilution of Kodak Kodafix solution in water for 5 minutes at 15° C. Sections were rinsed in water for 10 minutes, dried, stained with hematoxylin and eosin, and mounted with Permount.

EXAMPLE 12 Production of Transgenic Mice Expressing TGF-β₁ in the Epidermis

The pHK1 epidermal-specific targeting vector was also used to target a constituitively active form of TGF-β₁ to the epidermis of transgenic mice (Roche, et al., Inhibition of Skin Development by Over-expression of Transforming Growth Factor-β₁ in the Epidermis of Transgenic Mice, Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 5237-5241 (1993), incorporated herein by reference). These mice exhibited a severe skin phenotype resulting from complete suppression of epidermal cell proliferation. The skin of these animals was very shiny and tautly stretched, and this appeared to restrict their ability to move and breath; death occurred within 24 hours. Both of these studies document the profound phenotypic effects that result from deregulated expression of positive and negative growth factors in the epidermis of transgenic mice.

These results are in marked contrast to recent data generated via targeted disruption of the TGF-α and TGF-β₁ genes. Mice with a null mutations of the TGF-α gene exhibited a waviness of whiskers and fur and misalignment of hair follicles, but otherwise the skin appeared normal and wound healing was not impaired (Mann, et al., Mice with a Null Mutation of the TGF-α Gene Have Abnormal Skin Architecture, Wavy Hair, and Curly Whiskers and Often Develop Corneal Inflammation, Cell, Vol. 73, pp. 249-261 (1993); Luetteke, et al., TGF-α Deficiency Results in Hair Follicle and Eye Abnormalities in Targeted and Waved-1 Mice, Cell, Vol. 73, pp. 263-278 (1993)).

Although targeted disruption of the TGF-β₁ gene resulted in death at about 3-4 weeks after birth from multifocal inflammatory disease, no histological changes were detected in the skin (Shull, et al., Targeted Disruption of the Mouse Transforming Growth Factor-β1 Gene Results in Multifocal Inflammatory Disease, Nature, Vol. 359, pp. 693-699 (1992); Kulkarni, et al., Transforming Growth Factor β1 Null Mutations in Mice Causes Excessive Inflammatory Response and Early Death, Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 770-774 (1993)).

The failure of TGF-α and TGF-β₁ null mice to manifest severe skin phenotypes suggests that there may be functional redundancy among the ligands that bind EGF and TGF-β receptors in this tissue, and underscores the value of studies that utilize deregulated expression of these ligands in determining their role in normal and abnormal epidermis.

EXAMPLE 13 Production of Transgenic Mice Expressing the HIV tat Gene in the Epidermis

Patients infected with the human immunodeficiency virus (HIV) are at high risk for the development of specific AIDS-associated cutaneous disorders. Often patients manifesting symptoms have skin lesions ranging from hyperproliferative conditions such as psoriasis to Kaposi's sarcoma and metastatic basal cell carcinoma. The precise role of HIV genes, the cells of origin and hence etiology of such skin lesions remains unknown. It may be that specific HIV genes, e.g., the trans-regulatory protein tat, play a role directly or indirectly on the homeostatic mechanisms of host cells and tissues. Alternatively, the HIV tat gene may interact with or activate other viral genes present from latent or opportunistic infections, e.g., human papilloma virus (HPV). To directly assess the role of keratinocytes in the development of AIDS-associated cutaneous disorders, the HIV tat gene is targeted to the epidermis of transgenic mice. Targeting of the tat gene and exclusive expression in keratinocytes is achieved by the use of the human keratin K1 vector (FIG. 7). The development of strains of mice which develop cutaneous lesions with predictable kinetics as a result of expression of the HIV tat gene alone or in combination with other oncogenes serves as a useful model for assessing therapeutic potential of antisense pharmaceuticals designed to inhibit expression of the HIV tat gene.

EXAMPLE 14 Utilization of the HK1 Vector for Gene Therapy Applications

Where exclusive expression in epidermal cells is desirable and for transient expression the HK1 vector is an excellent choice for gene therapy. Unlike the human keratin K1 gene itself, the human keratin K1 vector derived from the 12 kb fragment is expressed in proliferating basal cells in the epidermis. In more recent transgenic experiments, it has been determined that a larger fragment containing the human keratin K1 gene, a 18 kb EcoRV fragment (Shown schematically in FIG. 8), is expressed exactly like the endogenous mouse K1 gene, i.e., post mitotically in cells committed to terminal differentiation. These cells are programmed to die and will eventually slough into the environment. Therefore, for human applications where transient expression is desired, it is possible to design a vector that will only be expressed in cells after they commit to terminal differentiation and begin moving upward toward the outer layers of the epidermis. The vector will be expressed approximately 10-14 days prior to being shed into the environment. This can be accomplished by inserting additional 5' flanking sequences from the 18 kb EcoRV fragment onto the end of the original human keratin K1 vector (See FIG. 9). Smaller portions of this 5' region can be chosen to provide this function using standard methodology (as for 3' regions).

EXAMPLE 15 Detection of Carcinogens and Tumor Promoters

Short-term tests (STTs) for genotoxic chemicals were originally developed as fast, inexpensive assays to assess the potential hazard of chemicals to humans. However, a recent report summarizing the results of a project initiated by the National Toxicology Program to evaluate the ability of STT's to predict rodent carcinogenicity questions the validity of relying solely on STT's. Three of the most potent carcinogens, detected in the rodent assays, produced no genetic toxicity in any of the four STTs evaluated (Tennant, et al., Science, Vol. 236, pp. 933-941 (1987)). Thus, to receive EPA/FDA approval for new compounds, chemical, agricultural, food and drug companies are currently required to perform two year animal tests costing up to $2 million. The development of new transgenic strains of mice that have been genetically engineered to rapidly detect carcinogens and tumor promoters would substantially reduce the overhead cost of long-term animal studies. The suitability of the transgenic mouse lines claimed in this patent application for rapid detection of carcinogens is initially determined with a known skin carcinogen, DMBA, to determine whether benign lesions appear earlier than in control non-treated litter mates. To determine suitability for detecting tumor promoters, a known promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) is applied to ras^(Ha), and fos mice. Since benign lesions in ras^(Ha) mice and hyperplasia in fos mice appeared at sites of wounding (i.e., tagged ears), and wounding can promote tumor formation, these lines are useful for these studies.

EXAMPLE 16 Cell Type and Differentiation-specific Regulatory Elements Controlling Expression of Keratin K1 Gene

The mammalian epidermis is composed of four histologically defined layers each of which represents a distinct stage of differentiation of the epidermal keratinocyte. Keratinocytes are the major cell type of the epidermis and arise from stem cells in the basal layer. Upon commitment to differentiation they lose their proliferative potential and migrate to the spinous layer. With further maturation they enter the granular layer and finally terminate as cornified squames in the stratum corneum before being sloughed into the environment. The degree of differentiation can also be defined biochemically by the expression of marker proteins that characterize each stage. For instance, basal keratinocytes express keratins K5 and K14 (Fuchs and Green, Cell, Vol. 19, pp. 1033-1042 (1980); Woodcock-Mitchell, et al., J. Cell. Biol., Vol. 95, pp. 580-588 (1982)). Once they enter the differentiation pathway to become spinous layer cells, they down regulate the genes for K5 and K14 and up regulate the genes for the differentiation-specific keratins, K1 and K10 (Roop, et al., Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 716-720 (1983); Schweizer, et al., Cell, Vol. 37, pp. 159-170 (1984); Reganier, et al., J. Invest. Dermatol., Vol. 87, pp. 472-476 (1986)). The expression of K1 precedes that of K10 and is one of the earliest events in keratinocyte differentiation. K1 can be observed in the occasional basal cell that has already ceased mitotic activity and is about to migrate into the spinous layer. Transcription of K1 and K10 is restricted to spinous layer cells and when these mature into granular layer cells, the genes for K1 and K10 are down regulated and other genes, notably loricrin and filaggrin are induced.

Both in vitro and in vivo studies have implicated calcium as a major modulator of epidermal differentiation. A calcium gradient has been identified in the epidermis with the basal and spinous layers having calcium levels much lower than that observed in serum and rising to much higher than serum levels in the upper granular layer and stratum corneum (Malmqvist, et al., Nucl. Instrum. Methods Phys. Res., Vol. 3, pp. 611-617 (1984); Menon, et al., J. Invest. Dermatol., Vol. 84, pp. 508-512 (1985)). In vitro studies have shown that keratinocytes maintain their proliferative capacity when calcium levels of the culture medium are kept below 0.1 mM and that differentiation ensues with calcium levels of 0.1 mM or higher (Hennings, et al., Cell, Vol. 19, pp. 245-254 (1980)). Moreover, differentiation-specific genes including keratins K1 and K10 can be induced in cultured keratinocytes by raising the level of calcium in the medium (Roop, et al., Differentiation, Vol. 35, pp. 143-150 (1987); Yuspa, et al., J. Cell Biol., Vol. 109, pp. 1207-1217 (1989)).

In contrast, retinoic acid appears to suppress epidermal differentiation and indeed, promotes an undifferentiated phenotype in keratinocytes in culture (Yuspa and Harris, Exp. Cell Res., Vol. 86, pp. 95-10517 (1974)). Expression of the differentiation-specific markers are also suppressed, including K1 and K10 (Roop, et al., Differentiation, Vol. 35, pp. 143-150 (1987); Fuchs and Green, Cell, Vol. 25, pp. 617-625 (1981)) loricrin (Hohl, et al., J. Invest. Dermatol., Vol. 96, pp. 414-418 (1991)) and filaggrin (Asselineau, et al., Differentiation, Vol. 45, pp. 221-229 (1990)). In addition, a retinoic acid gradient may also exist in the epidermis, with high levels in the basal layer declining to much lower levels in the differentiated layers (Vahlquist, et al., Upsala J. Med. Sci., Vol. 92, pp. 253-257 (1987)).

Another regulator of epidermal differentiation is the active metabolite of Vitamin D₃, 1,25 dihydroxyvitamin D₃. It has been shown that Vitamin D₃ is a potent inhibitor of proliferation (Matsumoto, et al., Biochem. Biophys. Res. Commun., Vol. 166, pp. 916-923 (1990)) and moreover, will promote the differentiation of spinous and granular cells into the corneocytes of the stratum corneum (Hosomi, et al., Endocrinology, Vol. 113, pp. 1950-1957 (1983); Regnier and Darmon, Differentiation, Vol. 47, pp. 173-188 (1991)). Vitamin D₃ is produced by keratinocytes and autoradiographic studies suggest a concentration of 1,25-dihydroxyvitamin D₃ in the suprabasal layers of the epidermis (Stumpf, et al., Cell Tissue Res., Vol. 238, pp. 489-496 (1984)). Until now, a direct role for Vitamin D₃ on epidermal keratin gene expression has not been reported (Blumenberg, et al., J. Invest. Dermatol., Vol. 98, pp. 42s-49s (1992)).

The best studied example of transcription factors that regulate the differentiation-specific genes of the epidermis is the HK1 gene. A 10.8 kb transgene, containing nucleotides -1246 to +9495 from HK1, was correctly regulated with respect to tissue and developmental specificity in transgenic mice (Roop, et al., in: The Biology of Wool and Hair (Rogers, G. E., Reis, P. J., Ward, K. A., and Marshall, R. C. eds), pp. 311-324, Chapman and Hall, New York (1988); Roop, et al., in: Pharmacology of Retinoids in the Skin (Reichert, U., and Shroot, B., eds.) pp. 1-7, S. Karger, Basal (1989); Rosenthal, et al., Cell Growth and Differentiation, Vol. 2, pp. 107-113 (1991)). Furthermore, the expression of HK1 in keratinocytes cultured from these mice could be induced by the addition of calcium to the medium (Rosenthal, et al., Cell Growth and Differentiation, Vol. 2, pp. 107-113 (1991)). These studies suggested that the transgene encodes all the cis-acting regulatory elements necessary to mediate the calcium response. Recently, an array of regulatory elements that respond to increased calcium levels in vitro were identified and found to reside within a 4.4 kb fragment, 3' of the HK1 gene (Huff, et al., J. Biol. Chem., Vol. 268, pp. 377-384 (1993)). The sequences that mediate the calcium response have recently been defined to a 249 bp fragment that lies 7.9 kb downstream of the HK1 promoter (Rothnagel, et al., J. Invest. Dermatol., Vol. 101, pp. 506-513 (1993)).

A detailed analysis of the 249 bp fragment that encodes the calcium response element (CaRE) was performed. Footprinting and mobility shift assays have identified adjacent cis-elements. One of these encodes an AP-1 element to which all of the calcium response can be attributed. The adjacent region encodes a hormone responsive element through which both vitamin D₃ and retinoic acid are able to modulate the calcium response.

Plasmids and Constructs

The plasmid pHIVLTR.CAT (Rosen, et al., Cell, Vol. 41, pp. 813-823 (1985)), was used as a control. Details on the generation of the 249 bp fragment that encodes the calcium response element (CaRE) have been given elsewhere (Rothnagel, et al., J. Invest. Dermatol., Vol. 101, pp. 506-513 (1993), hereby incorporated by reference). Briefly, the 249 bp (+7820 to +8069 (numbering of base pairs is with respect to the start of HK1 transcription)) fragment was generated by polymerase chain reaction and cloned using Bgl II and Bam HI restriction sites into the plasmid vector pSP72 (Promega, Madison, Wis.). The 249 bp fragment was subsequently self ligated into four tandem repeats and inserted into the Bgl II site of pA10CAT₂ in an antisense orientation relative to the CAT (chloramphenicol acetyl transferase) gene. This construct is referred to as p249^(b) in Rothnagel, et al., J. Invest. Dermatol., Vol. 101, pp. 506-513 (1993), and in this specification as CaRE.CAT. The constructs, CaREΔA.CAT and CaREΔB.CAT, were similarly constructed from CaREΔA and CaREΔB. These versions of the 249 bp fragment were created using polymerase chain reaction (Higuchi, in: PCR Technology, Principles and Applications for DNA Amplification (Erlich, H. A. ed) pp. 61-70, Stockton Press, New York (1989), to delete the entire protected region defined by footprint analysis. CaREΔA is lacking nucleotides +7895 to +7921 and CaREΔB is lacking nucleotides +7924 to +7948.

Cell Culture and Transfection

Primary murine keratinocytes were cultured and transfected as described in Harper, et al., J. Invest. Dermatol., Vol. 91, pp. 150-153 (1988). Five micrograms of plasmid DNA plus 25 μg carrier DNA were transfected by calcium phosphate in medium containing 0.1 mM potassium to block calcium induced differentiation Id. After 24 hours, the transfected cells were switched to medium containing 0.05 mM calcium and 24 hours later switched to media containing the appropriate concentrations of calcium, 12-0-tetradecanoylphorbol-13-acetate (TPA), or steroid hormones. Cells were harvested after 48 hours in these media and assayed for CAT activity (Gorman, et al., Mol. Cell. Biol., Vol. 2, pp. 1044-1051 (1982)). The CAT assays were normalized by protein content and activity visualized by autoradiography after separation by thin layer chromatography. These experiments were repeated at least three times.

Nuclear Extracts

Nuclear extracts were made from primary murine keratinocyte cultures and prepared essentially as described by Dignam, et al., Nucleic Acid Res., Vol. 11, pp. 1475-1489 (1983). The protein concentration of each extract was determined by the Bradford assay (Bradford, Anal Biochem., Vol. 72, pp. 248-254 (1976)). Aliquots of nuclear extracts were stored at -80° C.

Band Shift Assay

Purified fragments of either the intact CaRE, CaREΔA or CaREΔB were end-labeled at the Bgl II site with DNA polymerase Klenow fragment and ³² P!dATP (3,000 Ci/mmol). Each ³² P-labeled DNA fragment (10-20 fmol, 10⁵ cpm) was incubated with 2-4 μg nuclear protein extract and 3 μg nonspecific DNA poly (dI/dC) for 15 minutes at room temperature in 20 μl of binding buffer 20 mM HEPES (pH 7.8), 60 mM KCI, 2 mM dithiothreitol, 2.5 mM MgCl₂ and 2% Ficoll!. The mixtures were loaded directly onto a 4% polyacrylamide gel and electrophoresed at 30 mA at room temperature for 2 hours. The gels were dried and analyzed by autoradiography.

DNase 1 Footprinting

End labeled probes for footprinting were prepared as described above. Each labelled DNA probe (20-40 fmol) was incubated with 2-8 μg nuclear protein extract or one footprinting unit of purified c-Jun (Promega), 20 ng of poly (dI/dC) and binding buffer (see above) to a total reaction volume of 20 μl. After 15 minutes incubation at room temperature, the reaction was terminated by addition of 100 μl of stop solution containing 1 μg pBR322 plasmid DNA, 15 mM EDTA, 0.15% SDS and 1.5 μg of proteinase K. Samples were analyzed as described by Pastoricic, et al. (Pastoricic, et al., Mol. Cell. Biol., Vol. 6, pp. 2784-2791 (1986)).

Oligonucleotides

All oligonucleotides are double-stranded (only one strand is shown).

AP-1 (Promega) 5'-CGCTTGATGAGTCAGCCGGAA-3'

NS (NON SPECIFIC) 5'-GGCCTGGGGCTCTAGAGGAAGCCGG-3'

The following sequences are from Umesono, et al., Cell, Vol. 65, pp. 1255-1266 (1991):

VDRE (DR3) 5'-AGCTTCAGGTCAAGGAGGTCAGAGAGCT-3'

TRE (DR4) 5'-AGCTTCAGGTCACAGGAGGTCAGAGAGCT-3'

RARE (DR5) 5'-AGCTTCAGGTCACCAGGAGGTCAGAGAGCT-3'

Identification of Nuclear Protein-DNA Interactions Within the CaRE of HK1

Sequences within a 249 bp fragment located between +7820 to +8069 relative to the transcription start site of HK1 have been shown to direct transcription of both heterologous and homologous promoters in a cell-type specific manner in response to increased calcium levels (Huff, et al., J. Biol. Chem., Vol. 268, pp. 377-384 (1993); Rothnagel, et al., J. Invest. Dermatol., Vol. 101, pp. 506-513 (1993)). To define the sequences that are important for CaRE function, the factor binding sites within the 249 bp fragment were first identified. The CaRE bearing fragment was asymmetrically end-labeled and incubated with a keratinocyte nuclear extract in the presence of DNase I. Two protected regions were predominant. The 5' most footprint was designated as FP(A) and the adjacent footprint as FP(B). The protected region of FP(A) spans nucleotides +7895 to +7921 and contains within it an AP-1 consensus sequence, (TGATTCA) (Lee, et al., Nature, Vol. 325, pp. 368-372 (1987); Angel, et al., Cell, Vol. 49, pp. 729-739 (1987)).

An examination of the sequences protected in FP(B), which spans nucleotides +7924 to +7948, revealed an inverted palindromic sequence. This region contains two half-sites of the canonical hormone response element, (A/G)GGTCA (Evans, Science, Vol. 240, pp. 889-895 (1988)), separated by three nucleotides.

To confirm that binding to FP(A) is due to nuclear factor interaction with the AP-1 site, a 25 bp double-stranded oligonucleotide, containing the AP-1 consensus sequence, was added to the binding reaction. Binding to FP(A) was almost completely abolished by the addition of the competing oligonucleotide whereas that of FP(B) was unaffected. In contrast, the addition of 100 molar excess of a 25 bp nonspecific oligonucleotide had no effect on nuclear factor binding to either FP(A) or FP(B), whereas the addition of the unlabeled 249 bp CaRE fragment, inhibited protection of both regions. To test whether the protected region of FP(A) is due to the interactions of AP-1 transacting factors to this sequence, DNase I protection analysis was performed on the 249 bp fragment in the presence of purified c-Jun protein. As shown in FIG. 10, c-Jun binds to FP(A), although the boundary of the protected region is slightly different from that defined by the keratinocyte nuclear extract. Note that c-Jun did not interact with FP(B) nor with other potential cis-elements encoded by this fragment. The change in the footprinting pattern observed in the FP(B) region with the addition of c-Jun may be attributable to non-specific effects caused by the relatively high levels of c-Jun protein in this assay.

In mobility shift experiments on the CaRE encoding fragment three retarded complexes were consistently observed. Complex II disappeared upon the addition of 60-fold molar excess of an oligonucleotide containing the AP-1 consensus sequence, suggesting that this complex was formed by interaction of AP-1 factors to FP(A). Complexes I and III disappeared when an oligonucleotide encoding the thyroid hormone response element was used as the competitor, suggesting that proteins related to members of the thyroid receptor superfamily interact with cis-elements located within the FP(B) region.

These results were confirmed by repeating the gel shift assay on end-labeled fragments where the FP(A) and FP(B) regions were deleted individually from the original 249 bp CaRE encoding fragment. One complex (Complex II) was formed with a fragment lacking the FP(B) region (CaREΔB) and was specifically competed by the AP-1 oligonucleotide but not by oligonucleotides encoding the thyroid response element, retinoic acid response element, or Vitamin D₃ response element. Two complexes (Complexes I and III) were observed with a CaRE deletion mutant lacking the FP(A) region (CaREΔA) and both complexes were removed upon the addition of oligonucleotides encoding hormone response elements.

The stability of these complexes were unaffected by the addition of the AP-1 oligonucleotide. Taken together, these data suggest that members of the AP-1 family of transacting factors interact with FP(A) and that members of the steroid hormone superfamily can potentially interact with the FP(B) site.

Calcium Induction is Mediated Through the FP(A) Site

To assess whether the sequences protected in FP(A) and FP(B) were functionally involved in mediating the calcium response, a reporter construct was employed consisting of the CaRE linked to a SV40 minimal promoter CAT construct. The CaRE.CAT construct has been shown to be activated by increasing concentrations of calcium in the medium (Rothnagel, et al., J. Invest. Dermatol., Vol. 101, pp. 506-513 (1993)). To test whether endogenous AP-1 factors were able to induce CAT activity from the CaRE.CAT construct we added 12-0-tetradecanoylphorbol-13-acetate (TPA) to transfected keratinocytes. TPA is a potent inducer of AP-1 factors in keratinocytes (Dotto, et al., EMBO J., Vol. 5, pp. 2853-2857 (1986)), and is able to activate CaRE.CAT in a dose dependent manner. Thus both calcium and TPA are able to induce CAT activity from the CaRE.CAT construct.

To determine the contribution of each protected region to CaRE activity the fragments lacking either FP(A) or FP(B) were linked to pA10CAT₂ and their ability to induce reporter gene activity in the presence of calcium was tested. Deletion of FP(B) did not affect calcium, responsiveness while deletion of FP(A) completely abolished the calcium response. This observation suggests that all of the calcium response mediated by the CaRE can be attributed to factors interacting with the sequences protected in FP(A).

Vitamin D₃ and Retinoic Acid Modulate Calcium Induction of the HK1 CaRE

To determine the function of the FP(B) region, various steroid hormones, including thyroid hormone, retinoic acid, Vitamin D₃, and estrogen, were used to induce the activity of the 249 bp element in low calcium medium. These hormones by themselves were unable to induce CAT activity in a CaRE.CAT fusion construct. To determine whether these hormones were able to modulate calcium induction of the CaRE, transfected keratinocytes were treated with each hormone in the presence of high calcium medium (0.35 mM). Thyroid hormone, retinoic acid or estrogen had no effect on calcium induction but interestingly, Vitamin D₃ was found to suppress calcium induction. Since it has been observed that steroid hormone receptors are able to form heterodimers with other members of this superfamily (Glass, et al., Cell, Vol. 59, pp. 697-708 (1989)), it was asked whether the simultaneous addition of two hormones could influence CaRE activity in the presence of activating levels of calcium. To assess this, thyroid hormone, retinoic acid or estrogen were added along with Vitamin D₃ and calcium after transfection of the CaRE.CAT reporter construct. In combination, retinoic acid was able to reverse the suppression of calcium induction by vitamin D₃. Thyroid hormone and estrogen had no effect on Vitamin D₃ suppression of the CaRE.CAT construct.

In order to show that hormonal modulation of CAT activity was specific to the CaRE, the effects of Vitamin D₃ alone or in conjunction with retinoic acid on the activity of another TPA responsive promoter (pHIVLTR.CAT) in transfected keratinocytes was tested. The HIVLTR promoter is also inducible by calcium but its activity was only marginally suppressed by Vitamin D₃, in contrast to the CaRE of HK1. Moreover, the co-addition of retinoic acid had no effect on the activity of this promoter. To determine whether the modulation of CaRE activity by Vitamin D₃ and retinoic acid was mediated through the interaction of receptors with the hormone response element of FP(B), the reporter construct lacking FP(B) was tested for Vitamin D₃ suppression of calcium induction. In comparison with the CaRE.CAT construct, the calcium response of CaREΔB.CAT was less affected by Vitamin D₃ and interestingly, this suppression could be relieved by the co-addition of retinoic acid.

The above results are the first example of composite regulation of a keratin gene by AP-1/steroid receptor complexes. Interestingly, the HK1 promoter and immediate upstream sequences were unable to activate transcription of an HK1.CAT reporter fusion construct in the absence of the 3' CaRE. These results suggest that HK1 transcription requires elements both proximal and distal to the promoter and that the 3' CaRE element is functionally important for transcription of this gene. It is noteworthy that another keratin gene, HK14, was also found to require both proximal and distal elements for efficient transcription (Leask, et al., Genes Develop., Vol. 4, pp. 1985-1998 (1990)).

The proximal footprint, FP(A), encompasses sequences between +7895 and +7921 with respect to the start of HK1 transcription and is responsible for mediating the calcium activation of both homologous and heterologous promoters. This region encodes an AP-1 site and it was inferred from the footprint and gel shift assays that AP-1 factors bind to this region to confer the calcium response. A second potential AP-1 site within the 249 bp fragment was identified by sequence homology comparison in the earlier study by Huff, et al. (Huff, et al., J. Biol. Chem., Vol. 268, pp. 377-384 (1993)), but appears to be non-functional in keratinocytes. This site was not protected in footprinting assays by proteins from keratinocyte nuclear extracts nor by purified c-Jun. Moreover, the CaREΔA construct which lacks the first AP-1 site within FP(A) but retains the second potential AP-1 site, was unable to induce CAT expression. The two AP-1 sites differ only in two positions, one of which occurs at a position relatively insensitive to substitutions (Risse, et al., EMBO J., Vol. 8, pp. 3825-2832 (1989)). These results imply that AP-1 factors in keratinocytes can discriminate between these two sites and it is possible that sequences neighboring the AP-1 site within FP(A) also contribute to nuclear factor binding to this region (Ryseck and Rodrigo, Oncogene, Vol. 6, pp. 533-542 (1991)).

AP-1 sites have been identified in keratins K8 and K18 (Tamai, et al., Gene, Vol. 104, pp. 169-176 (1991); Oshima, et al., Genes Develop., Vol. 4, pp. 835-848 (1990)), as well as in the promoter regions of the keratinocyte-specific, human papilloma viruses types 16 and 18 (Cripe, et al., The New Biologist, Vol. 2, pp. 450-463 (1990); Offord and Beard, J. Virol., Vol. 64, pp. 4792-4798 (1990); Mack and Laimins, Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 9102-9106 (1991); Thierry, et al., J. Virol., Vol. 66, pp. 3740-3748 (1992)). An AP-1 site in the first intron of human K18 has been shown to be activated by co-expression of Fos and Jun (Oshima, et al., Genes Develop., Vol. 4, pp. 835-848 (1990)). Similarly, Fos and Jun have been shown to bind to the AP-1 sites within the long control region of human papilloma viruses where it is suggested that AP-1 sites are necessary but not sufficient for transcription. However, none of the above sites have been reported to be involved in calcium induction of these genes. In this respect the AP-1 site identified in the HK1 CaRE appears to be unique.

The expression of HK1 mRNA is tightly regulated and restricted to the spinous layer of the epidermis which can be considered as an intermediate state between the loss of proliferative potential and cell death. Since calcium can inhibit proliferation of keratinocytes and promote their differentiation and Vitamin D₃ can induce cornification of cells already undergoing differentiation, we propose that calcium promotes HK1 gene transcription through the upregulation of AP-1 factors and their subsequent binding to the FP(A) site, concomitant with or soon after the cell enters the differentiation pathway. HK1 transcription would then be maximal in the mature spinous layer cell where retinoic acid levels are high enough to antagonize the action of vitamin D₃ keeping the FP(A) site responsive to calcium activation. Later, as the keratinocyte differentiates into a granular layer cell, where Vitamin D₃ levels are maximal and retinoic acid levels are at their lowest, the activity of the FP(A) site would be suppressed and HK1 transcription down regulated. Thus the interaction of these modulators with the CaRE can account for the restricted expression of HK1 observed in vivo.

In summary, we have demonstrated the interaction of AP-1 factors with FP(A) and that of Vitamin D₃ and retinoic acid through their receptors with FP(B). The complex interplay between each of these factors serves to restrict expression of HK1 to the spinous cell during epidermal differentiation. We conclude therefore that the CaRE of HK1 functions as a differentiation stage-specific enhancer.

All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications which are incorporated herein by reference are incorporated to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The bioreactors, nucleic acid sequences, transformed epidermal cells, transgenic animals and human keratin K1 vector, along with the methods, procedures, treatments, molecules of specific compounds, are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     #       5) NUMBER OF SEQUENCES:     - (2) INFORMATION FOR SEQ ID NO:  1:     -      (i) SEQUENCE CHARACTERISTICS:     #            10747 base pairs     #              nucleic acid     #        singleTRANDEDNESS:     #           linearLOGY:     #       DNA (genomic)YPE:     -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  1:     - GAATTCGGCT GCTGTGCTGT CGTACAACAT GCTGTTTAGG ATCTTGCACA TG - #ATAGCTAG      60     - GTATTCTTGC TTCAAATCGC AGGCACCCCA CTTACCAACT GTGTAGACTT GA - #TCACGTTA     120     - TTCAACCCCT GTGTCTCTGC TTCCTCATTT TACAAATGGG GAGAAAAATA GC - #ATCTATCT     180     - CAAAGTTGTG AAAATTAAGC AAGTTAATAC ATATGTGCTA CGTAGAACAG TG - #CCTGGTAC     240     - ATGGTCAGTT TTTGATACAT GTTAGGTATT ATCATTATTA TCACCTCCAG AA - #ACAATTTA     300     - AACTTCTCAT ATAAGGCTCT CCAGACACCT CTCATTGTCT TCCCTTCCAA AT - #CTGCATTT     360     - ATCTCTCTCT CTTTGCAGTC CAGTGTGAGG CTTGAATCAC CTATCAAGCC TC - #ACCTCCAC     420     - CCCTGTGCTT TACAAAATGT CCTAGAGCTT CTATTTACTC GTCTCACTGC TC - #TGTGGGCT     480     - TTTTCACTCA AGGGCGTTTG CATGCTATCC ATTGCTACCT GTTTTCTGTT GC - #TGGTGTCT     540     - GTCTCCTGCT CTATCTTTGA AGAAAAGAAA CAAGAAAAGG AATAACTGAG AA - #ACAGAGAA     600     - AAAAAATGTC TCTCCCTTCT GGTTCTTCCA GACCACCCAC TCATCCATCT TG - #TTCAATGA     660     - CAGCTTCTCT TCCTTTAATT AATCACTGTG GTATATTTAT AAAGCTTATA TT - #TATGAAAG     720     - ACCTTTTAAT TTTTTAGTTA TTAAAGCCCT TTCTCTTTGT CAGGTTGTAA CT - #GAGTGAGC     780     - TCTGGAGTTT GGAAAGAAGA TCTTAGAAAT GGGCCAGAGA GCTCCTTCTG AG - #ATCCAAGC     840     - ACGGAGAATT GCACCTGCTG TGCATGGTAA GAGAGTGTGC TTGGTAGCTC AC - #AAGGGCAA     900     - GGTGAGAATA GAAACTTTCA TGCCTTTTTG ATGGGGGTTA TGAAATCCTA CC - #AAGAAACA     960     - CCAGGTATCA GATGTGGGGT CCTGTTTTCC CAAAGCCACA AATGCTTGAA GG - #AAGATCTT     1020     - GTGTGATAAA ATAATTACCA CATGAACCAA TCTTGCATGC ACAGCAATTT TG - #AGAGCCCA     1080     - TCCTGGGAGC TAGGTGTGTA GTGTTTATCG TATTGTTGAG GCTCGTAAAA AT - #CTTGTATG     1140     - GCTGCAGGCA AGCCAAACCC TTGACAGGCA CTGCATCTCC GCTGACTCTA GA - #AGACCAAG     1200     - CCCAATTTCT TCCCTGTATA TAAGGGGAAG TCTCTATGCT TGGGGTAGAG GA - #GTGTTTAG     1260     - CTCCTTCCCT TACTCTACCT TGCTCCTACT TTTCTCTAAG TCAACATGAG TC - #GACAGTTT     1320     - AGTTCCAGGT CTGGGTACCG AAGTGGAGGG GGCTTCAGCT CTGGCTCTGC TG - #GGATCATC     1380     - AACTACCAGC GCAGGACCAC CAGCAGCTCC ACACGCCGCA GTGGAGGAGG TG - #GTGGGAGA     1440     - TTTTCAAGCT GTGGTGGTGG TGGTGGTAGC TTTGGTGCTG GTGGTGGATT TG - #GAAGTCGG     1500     - AGTCTTGTTA ACCTTGGTGG CAGTAAAAGC ATCTCCATAA GTGTGGCTAG AG - #GAGGTGGA     1560     - CGTGGTAGTG GCTTTGGTGG TGGTTATGGT GGTGGTGGCT TTGGTGGTGG TG - #GCTTTGGT     1620     - GGTGGTGGCT TTGGTGGAGG TGGCATTGGG GGTGGTGGCT TTGGTGGTTT TG - #GCAGTGGT     1680     - GGTGGTGGTT TTGGTGGAGG TGGCTTTGGG GGTGGTGGAT ATGGGGGTGG TT - #ATGGTCCT     1740     - GTCTGCCCTC CTGGTGGCAT ACAAGAAGTC ACTATCAACC AGAGCCTTCT TC - #AGCCCCTC     1800     - AATGTGGAGA TTGACCCTGA GATCCAAAAG GTGAAGTCTC GAGAAAGGGA GC - #AAATCAAG     1860     - TCACTCAACA ACCAATTTGC CTCCTTCATT GACAAGGTGA GTTTCTCTCT CA - #TTGCACTG     1920     - GTAGGGCTGC CGCTGGTCCA CTTGGGATTG GTGCAGTCAA AACACATGTA GG - #TTTGAACC     1980     - TCAAGTTTCC ATGTTTACAT GATTAAAAGG ATGTTTTGTG GAATGGTCTC CT - #AGGAGATA     2040     - TGTTAGATGT ATGCTTGTGA ATGGTGTTAA TGACTCTCTC TTTGACAAAG GG - #TTCGTGGT     2100     - CGACCTAAAG GTGGGTCAGT GTGACATTAA CATTTAAGTG CTTTTTATTC AG - #CTCTTGAG     2160     - CGGAATTGGG ACTCATATCT GTTGAATGAA GATAATAGAA ATGGGGCTAA CT - #GAACTTTC     2220     - CAGGGTGCAA GTGAGAACCC TGGAAAGGTC TTCCTAACCA TAGAAAGGGA GT - #TGAGTGTG     2280     - AACATAGTAT AGAGTGTTAT TGTAGCAGAA AACATGTGGT CAGTCAGTGC CA - #AACATCTT     2340     - TTGCTGTCAG AGGGGAGCTC TGCCTTCTAA TAATTTTACA TTGGTACTGG AT - #GAGGCTAG     2400     - AGTTTTTTTA TACTAATATC TCCAAAAATC AGCTCTAAAA AACTCAGATA AA - #CCATTTTT     2460     - TTAATTTTTT GCTTAATCAT TAATAGTGCC AATCCAAGGT TATCCACAAC AA - #ATTTCAAA     2520     - TCCAATTTTG AATTTTCCTG ATATACTTTT GAAATGTGTG TGTGTCCTGG GG - #ATGCAAAC     2580     - CAGTTTTTAT GGTAATATAC CTAACAAAAT TTTGGAAGGC AAATCTCTTA AA - #TACCATGC     2640     - ACCTATTTCA AAACATAATT GCAATAATTC TGTATGCGCT TTGCTATTGG TA - #TTTGTTTA     2700     - GTTACTCCCT TCCAAGCCCT CTCTGAATTA ACAAGTTGGG TTTTATTATG CA - #GATGATAT     2760     - TAACTTGATC ATCTTCTTCC TATTTCTCTG TCATGGTCAG AAGATAGGAA TT - #GAGGTTCT     2820     - TTTCCAAATG AGGCACAGTT CTCCATGGCT ATGAGACTCC ATTTATGCAT CA - #GGAGTAAA     2880     - GGGGTCTTGT GTTTTTAGGT GAGGTTCCTG GAGCAGCAGA ACCAGGTACT GC - #AAACAAAA     2940     - TGGGAGCTGC TGCAGCAGGT AGATACCTCC ACTAGAACCC ATAATTTAGA GC - #CCTACTTT     3000     - GAGTCATTCA TCAACAATCT CCGAAGGAGA GTGGACCAAC TGAAGAGTGA TC - #AATCTCGG     3060     - TTGGATTCGG AACTGAAGAA CATGCAGGAC ATGGTGGAGG ATTACCGGAA CA - #AGTAAGGG     3120     - ACCCTGTCTG GGCAGTTCTT AACTTTTGCT GTAAAAGAGT TCCAGAAAGT AA - #TAAGCTAA     3180     - GATCATGAAG CAGCATGTAG CTATGTCTTT TCTTAGGTTA GAGGCACATC AG - #TTTGACAT     3240     - TTTCAGAAAT CTTCATTTTC TCAGGAGATG GAAATAGTCT AGTGGTTTTA TT - #GCTCAGTA     3300     - GAAAGTAGTG GCCAATATGT CCTAGGTTCA TAATAGAAAG GCAGTGATAG GC - #AATGCCAC     3360     - CTTTAGTTTA GAATGCTGGA CTTCAGGTCT TACCACCTCT GAATCTCCTA AT - #TGTTTCTG     3420     - CTTTCCTGCA GGTATGAGGA TGAAATCAAC AAGCGGACAA ATGCAGAGAA TG - #AATTTGTG     3480     - ACCATCAAGA AGGTAAGCAA ATTCTGTAGG ACGGAACTCA CATTTGAAAT AA - #ATAAGGGA     3540     - AGAGGGTCTC CAATTACTAA GCAGAAAGCA GCCATGATAT GGAGAGCCAG GT - #AGTAGACC     3600     - TGGGGAGTAT ATGGAGTGGG GCTATATTTT TCACATCATC ATGGACCTGG AC - #TGATCCAG     3660     - GCACTTGGCT TCTCCATATT TCCCAGCACC TTACATAGTA AGTGGAGTGG CA - #GATTCTCA     3720     - GCAAGCCAGG CACACTCCCT TGATGGTGCT ATCCGGGGGT GGGACAGTTA GG - #GAACTGTG     3780     - ATTTACCTGG GGCAAAAAGG AGTGGAGTAG ACCCAAAGCT CCTTTTTTTG CT - #TGGAGAAT     3840     - CCCCTCACAG GTAATGAGAG GGACCTGCCC TGGAGAGAAC GTGCCTTCAT GA - #TGTCCCTT     3900     - GTTCCTCTAG GATGTGGATG GTGCTTATAT GACCAAGGTG GACCTTCAGG CC - #AAACTTGA     3960     - CAACTTGCAG CAGGAAATTG ATTTCCTTAC AGCACTCTAC CAAGCAGTAA GT - #CTTCCAGT     4020     - TTCAACCAAG TTTATCTAAA TGGAGAGTTT TTAAGCCGGA ACCCACAACG AT - #TCAGAAGA     4080     - ATAGATATTT ATCTTTTATT TCCTGACTGC TTTCTCTGTC TAAGTTGTTT TT - #TGTTTTAG     4140     - TGCTGTAAGA GTCACTAACC TATTATGTCT TGCAGGAGTT GTCTCAGATG CA - #GACTCAAA     4200     - TCAGTGAAAC TAATGTCATC CTCTCTATGG ACAACAACCG CAGTCTCGAC CT - #GGACAGCA     4260     - TCATTGCTGA GGTCAAGGCC CAGTACGAGG ATATAGCCCA GAAGAGCAAA GC - #TGAGGCCG     4320     - AGTCCTTGTA CCAGAGCAAG GTGAGTGGGC TGAAACCGTA GCCAGTTTCC CT - #GAAATGGC     4380     - TTGTCTTGCT ATCCTGTGTT ATCTCATGTA TGTGTGCCTG TGCCATGCTG AG - #TTCTGCCT     4440     - ACATTTAACA AACGCTATCT ACCATCTTTA GTATGAAGAG CTGCAGATCA CT - #GCTGGCAG     4500     - ACATGGGGAT AGTGTGAGAA ATTCAAAGAT AGAAATTTCT GAGCTGAATC GT - #GTGATCCA     4560     - GAGACTTAGA TCTGAAATCG ACAATGTCAA GAAGCAGGTA TGTGCTTTCT CC - #TTCTACCA     4620     - CTCAGCTGTA TGGAATGGGG GTAACCCTCA GGTAAAGGGC GAGTGCTTTC CT - #AGTTTTGA     4680     - ATCTTGCAAT TCAGCCCAAG GCTACATTAT TAGCCCTGGT TCCTTTTCTG AC - #TATGCTAG     4740     - TTTCCAGAAT GCAGCCATCA TGCTGGGTTC TCTTTAGGGA AATCTGTGAG AA - #TGGCCTAG     4800     - TAGAGAAAGA TGGGATGGTC AATGTGAGTG ATCTAGCCTA TGACCCAAAG TG - #GACTTAAG     4860     - AGTTGGGGAG TGAGAGGAAG GGCAGCCAGG AGGTTTTAGA GTAGGTGTTT AG - #AAGAATGT     4920     - CAAGTCTGTA AGGGTTGTAG GAGCCTTGAC TCAGGGCCAA GAGAGGCTGT TG - #AGTTATCC     4980     - CTAAGGTCTT TTAAGGAAGT CAACATGGTG ATGTGTTATC TGGAGGTGGG TG - #TGAGATGA     5040     - CTTAAGGCCA AGTGGTTCTG TTGGACTCAT TATTGGCCTC ACTGGAGTGG GG - #AGACCAAT     5100     - TGGGATGAGG AGGCCTAGTG GGGAATGCAT ATTATGAGAG GGTGTCATAT CT - #TTTTCAGA     5160     - TCTCCAACTT GCAGCAGTCC ATCAGTGATG CAGAGCAGCG TGGCGAGAAT GC - #CCTCAAGG     5220     - ATGCCAAGAA CAAGCTGAAT GACCTGGAGG ATGCCCTGCA GCAGGCCAAG GA - #AGACCTGG     5280     - CCCGCCTGCT GCGTGACTAC CAGGAGCTGA TGAACACCAA GCTGGCCCTG GA - #TCTGGAGA     5340     - TTGCCACCTA CAGGACCCTC CTGGAGGGAG AAGAAAGCAG GTGAGGAAGG GA - #CGCTGGGA     5400     - GTCGAACCTC TTCTCATGGT CTTCCTTCCT TGCAAGCTGA TTGTTGTTGA AG - #ATGCAGCC     5460     - ATCTGATTGC AGCTTGTGCT GGGTATGGGG AAATGAAAAG TACACGGAGC AG - #GAGGAAGG     5520     - ACCTAGTTTT ACTTTGGGAG CTGGAGTCCC AAGCTGTTTA TTTTTTTCTT CT - #AGGGCTGT     5580     - AACATATCTA GAAAGAGCTT TGAGGTGGAG CAAATTATTC TTTATCTGGG CT - #GCCTCAGA     5640     - TGGCAGCTGG CCTAAAGTCG GCATCTTTAG AGGGGGCCTT CATTGGCTGC AA - #GGCTCGTC     5700     - TCGTTTATAT GGGAATTTCT CCGTGTTTGT ACTCTTGCTG AGAAAAAATG AC - #AGGTCTGG     5760     - GAGGCCAGAG GGGATTGGAT TAAGTTTCAG ATTAAGTGCA TTGGAGAAGA CC - #CAGATGGG     5820     - GAAAGTCTTC AAGGTGGTGG AGCGGGGAAT GGGGAAGCGG TTTGGGAAGC TG - #GAGTGTCC     5880     - TGAGGAATTT TCTTATTTTC TCCTACAGGA TGTCTGGAGA ATGTGCCCCG AA - #CGTGAGTG     5940     - TGTGTAAGTA CAAGTCGATT TCTCAGGGGC ATGTGCAGGC TTTGTTGGGC TG - #GAAACGGA     6000     - GTTGAGGTTG AAAATAACTG AGCTTCCTCT TGCAGCTGTG AGCACAAGCC AC - #ACCACCAT     6060     - CAGTGGAGGT GGCAGCCGAG GAGGTGGCGG CGGTGGCTAC GGCTCTGGAG GT - #AGCAGCTA     6120     - TGGCTCCGGA GGTGGTAGCT ATGGTTCTGG AGGTGGCGGC GGCGGCGGCC GT - #GGCAGCTA     6180     - TGGCTCCGGA GGTGGCAGCT ATGGCTCTGG AGGTGGCGGC GGCGGCCATG GC - #AGCTACGG     6240     - CTCCGGAAGC AGCAGTGGGG GCTACAGAGG TGGCTCTGGA GGCGGCGGCG GC - #GGCAGCTC     6300     - TGGCGGCCGG GGCTCTGGCG GCGGGAGCTC TGGAGGCTCC ATAGGAGGCC GG - #GGATCCAG     6360     - CTCTGGGGGT GTCAAGTCCT CTGGTGGCAG TTCCAGCGTG AAGTTTGTTT CT - #ACCACTTA     6420     - TTCCGGAGTA ACCAGATAAA GAGATGCCCT CTGTTTCATT AGCTCTAGTT CT - #CCCCCAGC     6480     - ATCACTAACA AATATGCTTG GCAAGACCGA GGTCGATTTG TCCCAGCCTT AC - #CGGAGAAA     6540     - AGAGCTATGG TTAGTTACAC TAGCTCATCC TATTCCCCCA GCTCTTTCTT TT - #CTGCTGTT     6600     - TCCCAATGAA GTTTTCAGAT CAGTGGCAAT CTCAGTCCCC TGGCTATGAC CC - #TGCTTTGT     6660     - TCTTTCCCTG AGAAACAGTT CAGCAGTGAC CACCACCCAC ATGACATTTC AA - #AGCACCTC     6720     - CTTAAGCCAG CCAGAGTAGG ACCAGTTAGA CCCAGGGTGT GGACAGCTCC TT - #AGCATCTT     6780     - ATCTCTGTGC TGTTTTGGTT TTGTACATAA GGTGTAAGCA AGTTGTTTTT CT - #TTTGTGGA     6840     - GAGGTCTTAA ACTCCCCATT TCCTTGTTTT GCTGCAATAA ACTGCATTTG AA - #ATTCTCCA     6900     - TGTCTCGATC GCCCTTGTTT ACGGCACTGT CTAACCTGGA TGGGTGTTTT GT - #GAGGTAAA     6960     - AGAAGACACT AGAGCCACAT GGCATATGGG AAAGTCATGC ACACAAACAT GA - #GAAAAATG     7020     - CAGAGGCCAA CCAGGCAACA TTTCACCAGA CTGGAATCAC AGAGAGAGCA AG - #CACTTTCC     7080     - CAGATGGTGG GGATGTCATG GAGAAATGGA GAGACCGGGT GACAGGTTTT GT - #TCATTTGA     7140     - GAAGGCTTTC TTGAAAAGGG CAGTGAGCAA GCAGGTTGGG AGGAAGAGGT GT - #GGCATTGA     7200     - GAAGAAGGGA AAGTATTGCA TGAAAAAGTA ATTCTTCACG TGGAACAGCC AG - #TAAGGAGG     7260     - GGCATGAGTA ATATAGGGTC AGCAGTTACT GGAGCCAGAA TACAGACTTT GG - #CCTGGGGA     7320     - GTTCAAGAAC TAAGAGTGGT AATAGAGAGT TGGATATTCC ATTTCCCTTC TC - #TTTTTGTG     7380     - CCACCACCCA AAGCTCTGCA TAATCTAAGA AGTTCCCTTG TTGACACATA GC - #TCATACTT     7440     - GTGAAGTTGT ACAACAGGAT AGCATAGTGG CCAGAAGCAT GGACAGTTGA AC - #TCAGATAT     7500     - GCTTGGGTTT GAATCTTACC ATCACCATTT ACTAGTTCTG TAATACAGTG CA - #AGTTACAG     7560     - ACATCTCTGC ACCTCAGTTT TAGTATGTCT AAATTGGGGA TGATAATGCC TT - #CCTTGTGG     7620     - GGATAGTGTG AGGATTGAAT AAGATGAATA CACATGGCTG AGCACACAGC AA - #GCACTAAA     7680     - TAAGTGCCAG TTTTAATGAT AACGGTGATG ATGATGATGA TGATGATGAT GA - #CGTAACAT     7740     - TGCTTGTGGG ACTCCATACA GCTCAGTAGA TGCTTGCTCA AAGAAGCAAG TT - #ACCAAAAT     7800     - TTTTGTAATG GTTCTATGAA CGTGAAAAAA GCAGTCAACT TCTCTGAGGA TC - #AATTTCCT     7860     - TAGTTTCCAA TTAGGAAAAG TCTTCTTAGC TCCAGAGTCC CACAGGGCTA AT - #GGAATAAG     7920     - GAGAGGATAG ATCACACATG TATTATGCAA ACACAACTCA GGTGAGCTCT AT - #TCTTCCTT     7980     - CTCAGTTATC CCTTCTGTAG GGACCCCAGT GTCCCCTGCT GTCTTTCTGT GT - #CCTGACCG     8040     - GGAAACACAG TGTGCCTTGT CTACTCCATC ACTTGGCCAG CTGCATGCTT TC - #CTTTGCAG     8100     - GCTTGAAGCA AAGCTGGGTC TCGGACATTC TCAGGCACTG ACAAAGCTGT TT - #AGTTGTTG     8160     - CTGGGAAACA CTGGGAAATA GCCCTTTTGT TAAACACACA GAAACTAGCC TT - #CGCCCTGA     8220     - GCCAAATTCC TTAAACTCGT CTATGAAATT CCATAACCTG ACTCCTTAAC TG - #CAGACATA     8280     - CCCAGCTAGA ACATCCCTCA TGTCCCTGTC CACCGTGAGA ATGCTGCACT TC - #ACTCTGAA     8340     - CCTTTAGTCC TCCTTTTAAA TACTGCACAC TGATCACCCT GGTGTTTAGT GC - #TTTGTTTT     8400     - TTGGAATCCC ACCTGGCTCC ATTTTGGGAT GGTTCCGGGC ACTTCCCTAT GG - #AAATTCCC     8460     - CTGCTGTCAC TGTCAGAGTG AGTCCAGCAG TGGGTTTAGC TGGATGAAAC AC - #CACCATGT     8520     - CCATTTCCAT TCAGACTAAT GTCAGAATTT GAAAGGCACT ATGGTAGAGT AG - #AAAGAACA     8580     - AGGAACTGTA CTATTTAAAG GGCAGGCAAA GAAAAGGCAT CTATAGCTTA TA - #AGATGTGT     8640     - GGATCTTTGG ATGTGACTTG GCCATCCTGA GCCTAAGTTG TCTTGTAGGA GA - #AATGGGAA     8700     - TGAGAATATT TTCCTCTAGA CATCAAGAGG AAAAGAAATA TAACGTGAAA AC - #CTTTGTGA     8760     - ATTGTGAATG TGTTATACAG AGTAGCTAAA AGAATTAAAA AGGGAGTGAC AA - #AAAAGTAA     8820     - AAGGCAGCTG GCTGCTCAGG GCCTCCATGG AGGGAAGTAC CTTGATATGG TC - #ACTGTGGC     8880     - TCAGTGACAG CTCTGCAGGG ACAGGAAATT GATTTGTTAG TGCACCCAAA GT - #TGAATCTG     8940     - CTCCTGAGTA CTGATTTATG GGAACCAAAC ACACAAGAGA TGAAGGATGT GT - #CAACCAGA     9000     - ATGTCCAGCA TTAGCTTGTG GGGAAACACA TACTTCCAGT GACTGAAATA CC - #ATCCTGTT     9060     - ATCAAGAGAT CTGGGAAACT AAAGTACTGA CAAGAGCTGG CTTGATCTGT GG - #ATTTAGAA     9120     - CAATGAGAGT TAGGTGGCCT TGAGGGAGAT GATTCACTCT CCTTCACAGA AG - #AGCTGACC     9180     - TCTGGGGTCA ACAGATATAG CACCTCTTTC CCAGGGACGC TACTGAATGA AC - #AGTGATGT     9240     - GTTCTTATAC TCTGGCCCAG ATTTTCTACA TACTTTCTTA GGTTACAACT TT - #ATTTAGTC     9300     - ACATTTCAGT ACTGGGGATA CTCCTGTTTA TCTTCTTTGG ACTCGAGTTT TT - #ATGGGAAG     9360     - GTCATGAAAC AGAGAAAAAT ACAATTTGCA GGGAAACTTA CCAAGGCTTG TA - #AGGTTACA     9420     - AGGATTAAAT GAAAACCCTG TGTAAGTCAG TATATAGTGA AGAAGTAAAT TG - #AGTTAGAC     9480     - CAAACGCCAA AATGCATCCG CATTAGAAAG ACGATAAAGG AAGACTCTGG AT - #TCAGTTCT     9540     - GTTCAAAAAA CATTTTCTGC ACAAATACTA TGTATGAGGA ACTGGGCGTT GG - #GGAGATGA     9600     - TGATGAGTGA GACATGGTTC TTGCTTTCAG AGAGCCTAGA GACCTGGGTG GT - #AGCAATGG     9660     - TAGAGATACA TCCAAGACAC AGAAATAGAT ATACAGGAAC ACAGATGATT GA - #AAGTGATG     9720     - CTTGGCAGGG CTTTAAAGAA TGAATCAGAG TTTTTCAGGC AGACGAGGAT CT - #TCAAGGCA     9780     - GAGGGAATCA TATAGATAAG GACATAGAAG AGTGAAATTT CATGAAGTAG TT - #AAGCATCT     9840     - GAAGAAGCAT GGAATTAGTG ACAAGAAATG ATGCGGAAAA GATATCCAGA TC - #CAATCAAG     9900     - AAGGGCCTTG TTGGCATTCT ATGGAGTCTG GACTTTGGCT TCTGGGTCAC AA - #GTTCTCAG     9960     - ATGGGGTTTT CATATCTATT ATTAGACCTA CTATGTACTG GTCCAGTGGA AG - #GGAAAGGG     10020     - GTTGTCTTAC TGCTAGTGGA GTAGGAATTG GGTATGGACC ACAGCTTGTC TT - #GTTTCCAA     10080     - GTATTCCCTA AGAAATCTGG TCTGCTGATG GGAGATCTAT TTATGGAAAT GT - #CTTTTTCC     10140     - CTCAGGAATT TTATGTCGGA AACAGCTGTC ATAGGTGAGG AGGAACTGGT AA - #AAGTACTT     10200     - AATAGGAGAG TGTCATGGTC AGATTGGTGT TTTGGAAAAG TCAGCCAGGG CA - #GATTGGAG     10260     - AGGTCCATAT TGGAGGCAGG AAGACTTAAG AGACTATTGC AAAGGTGAAG AC - #AAAAGACG     10320     - ATAGGGACTT GCACTTTAAT TCCAGCCCTT AGAAGTAGTA GAAGGTCAGA AA - #TGAGAATA     10380     - TGCATTACAG AGATAGTTAG TTGCTATATC ATTAGGACTT GGTGATAGAT TG - #GATGAGGA     10440     - TGCGGTTGGG TGAGGCAAAG AGGAGAGTCC ACATTCCTGG TCTGGGTAGT AA - #CAAAGAAT     10500     - CTAGCAAGAG GGCTTGTGGG GAAAGATGCT GAGTTACGTA GCAAGTGCAT CT - #GCTTTATC     10560     - CTTGTAATGA ATGGGGCTAA AGGTGTAAAC CAAAGAGTCA TCAGCATTTG GA - #GGGTAGAA     10620     - TAAATCATCA GATAACTCAG GAAGAAGGAG CAGAAGAATT ACTGATACTC CC - #TGGAAGGA     10680     - AAACCGGAAG TAAATGGGAG AAACTTGCTC AAGTGGACAA AGTTTAACAG AC - #ATGAAGCA     10740     #      10747     - (2) INFORMATION FOR SEQ ID NO:  2:     -      (i) SEQUENCE CHARACTERISTICS:     #            6693 base pairs     #              nucleic acid     #        singleTRANDEDNESS:     #           linearLOGY:     #       DNA (genomic)YPE:     -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  2:     - GAATTCGGCT GCTGTGCTGT CGTACAACAT GCTGTTTAGG ATCTTGCACA TG - #ATAGCTAG      60     - GTATTCTTGC TTCAAATCGC AGGCACCCCA CTTACCAACT GTGTAGACTT GA - #TCACGTTA     120     - TTCAACCCCT GTGTCTCTGC TTCCTCATTT TACAAATGGG GAGAAAAATA GC - #ATCTATCT     180     - CAAAGTTGTG AAAATTAAGC AAGTTAATAC ATATGTGCTA CGTAGAACAG TG - #CCTGGTAC     240     - ATGGTCAGTT TTTGATACAT GTTAGGTATT ATCATTATTA TCACCTCCAG AA - #ACAATTTA     300     - AACTTCTCAT ATAAGGCTCT CCAGACACCT CTCATTGTCT TCCCTTCCAA AT - #CTGCATTT     360     - ATCTCTCTCT CTTTGCAGTC CAGTGTGAGG CTTGAATCAC CTATCAAGCC TC - #ACCTCCAC     420     - CCCTGTGCTT TACAAAATGT CCTAGAGCTT CTATTTACTC GTCTCACTGC TC - #TGTGGGCT     480     - TTTTCACTCA AGGGCGTTTG CATGCTATCC ATTGCTACCT GTTTTCTGTT GC - #TGGTGTCT     540     - GTCTCCTGCT CTATCTTTGA AGAAAAGAAA CAAGAAAAGG AATAACTGAG AA - #ACAGAGAA     600     - AAAAAATGTC TCTCCCTTCT GGTTCTTCCA GACCACCCAC TCATCCATCT TG - #TTCAATGA     660     - CAGCTTCTCT TCCTTTAATT AATCACTGTG GTATATTTAT AAAGCTTATA TT - #TATGAAAG     720     - ACCTTTTAAT TTTTTAGTTA TTAAAGCCCT TTCTCTTTGT CAGGTTGTAA CT - #GAGTGAGC     780     - TCTGGAGTTT GGAAAGAAGA TCTTAGAAAT GGGCCAGAGA GCTCCTTCTG AG - #ATCCAAGC     840     - ACGGAGAATT GCACCTGCTG TGCATGGTAA GAGAGTGTGC TTGGTAGCTC AC - #AAGGGCAA     900     - GGTGAGAATA GAAACTTTCA TGCCTTTTTG ATGGGGGTTA TGAAATCCTA CC - #AAGAAACA     960     - CCAGGTATCA GATGTGGGGT CCTGTTTTCC CAAAGCCACA AATGCTTGAA GG - #AAGATCTT     1020     - GTGTGATAAA ATAATTACCA CATGAACCAA TCTTGCATGC ACAGCAATTT TG - #AGAGCCCA     1080     - TCCTGGGAGC TAGGTGTGTA GTGTTTATCG TATTGTTGAG GCTCGTAAAA AT - #CTTGTATG     1140     - GCTGCAGGCA AGCCAAACCC TTGACAGGCA CTGCATCTCC GCTGACTCTA GA - #AGACCAAG     1200     - CCCAATTTCT TCCCTGTATA TAAGGGGAAG TCTCTATGCT TGGGGTAGAG GA - #GTGTTTAG     1260     - CTCCTTCCCT TACTCTACCT TGCTCCTACT TTTCTCTAAG TCAACATCGA AT - #TTGCCTCC     1320     - TTCATTGACA AGGTGAGTTT CTCTCTCATT GCACTGGTAG GGCTGCCGCT GG - #TCCACTTG     1380     - GGATTGGTGC AGTCAAAACA CATGTAGGTT TGAACCTCAA GTTTCCATGT TT - #ACATGATT     1440     - AAAAGGATGT TTTGTGGAAT GGTCTCCTAG GAGATATGTT AGATGTATGC TT - #GTGAATGG     1500     - TGTTAATGAC TCTCTCTTTG ACAAAGGGTT CGTGGTCGAC CTAAAGGTGG GT - #CAGTGTGA     1560     - CATTAACATT TAAGTGCTTT TTATTCAGCT CTTGAGCGGA ATTGGGACTC AT - #ATCTGTTG     1620     - AATGAAGATA ATAGAAATGG GGCTAACTGA ACTTTCCAGG GTGCAAGTGA GA - #ACCCTGGA     1680     - AAGGTCTTCC TAACCATAGA AAGGGAGTTG AGTGTGAACA TAGTATAGAG TG - #TTATTGTA     1740     - GCAGAAAACA TGTGGTCAGT CAGTGCCAAA CATCTTTTGC TGTCAGAGGG GA - #GCTCTGCC     1800     - TTCTAATAAT TTTACATTGG TACTGGATGA GGCTAGAGTT TTTTTATACT AA - #TATCTCCA     1860     - AAAATCAGCT CTAAAAAACT CAGATAAACC ATTTTTTTAA TTTTTTGCTT AA - #TCATTAAT     1920     - AGTGCCAATC CAAGGTTATC CACAACAAAT TTCAAATCCA ATTTTGAATT TT - #CCTGATAT     1980     - ACTTTTGAAA TGTGTGTGTG TCCTGGGGAT GCAAACCAGT TTTTATGGTA AT - #ATACCTAA     2040     - CAAAATTTTG GAAGGCAAAT CTCTTAAATA CCATGCACCT ATTTCAAAAC AT - #AATTGCAA     2100     - TAATTCTGTA TGCGCTTTGC TATTGGTATT TGTTTAGTTA CTCCCTTCCA AG - #CCCTCTCT     2160     - GAATTAACAA GTTGGGTTTT ATTATGCAGA TGATATTAAC TTGATCATCT TC - #TTCCTATT     2220     - TCTCTGTCAT GGTCAGAAGA TAGGAATTGA GGTTCTTTTC CAAATGAGGC AC - #AGTTCTCC     2280     - ATGGCTATGA GACTCCATTT ATGCATCAGG AGTAAAGGGG TCTTGTGTTT TT - #AGGTGAGG     2340     - TTCCTGGAGC AGGATCCCGG GTACCGCGGC CGCATCGATT CGATAAGAGA TG - #CCCTCTGT     2400     - TTCATTAGCT CTAGTTCTCC CCCAGCATCA CTAACAAATA TGCTTGGCAA GA - #CCGAGGTC     2460     - GATTTGTCCC AGCCTTACCG GAGAAAAGAG CTATGGTTAG TTACACTAGC TC - #ATCCTATT     2520     - CCCCCAGCTC TTTCTTTTCT GCTGTTTCCC AATGAAGTTT TCAGATCAGT GG - #CAATCTCA     2580     - GTCCCCTGGC TATGACCCTG CTTTGTTCTT TCCCTGAGAA ACAGTTCAGC AG - #TGACCACC     2640     - ACCCACATGA CATTTCAAAG CACCTCCTTA AGCCAGCCAG AGTAGGACCA GT - #TAGACCCA     2700     - GGGTGTGGAC AGCTCCTTAG CATCTTATCT CTGTGCTGTT TTGGTTTTGT AC - #ATAAGGTG     2760     - TAAGCAAGTT GTTTTTCTTT TGTGGAGAGG TCTTAAACTC CCCATTTCCT TG - #TTTTGCTG     2820     - CAATAAACTG CATTTGAAAT TCTCCATGTC TCGATCGCCC TTGTTTACGG CA - #CTGTCTAA     2880     - CCTGGATGGG TGTTTTGTGA GGTAAAAGAA GACACTAGAG CCACATGGCA TA - #TGGGAAAG     2940     - TCATGCACAC AAACATGAGA AAAATGCAGA GGCCAACCAG GCAACATTTC AC - #CAGACTGG     3000     - AATCACAGAG AGAGCAAGCA CTTTCCCAGA TGGTGGGGAT GTCATGGAGA AA - #TGGAGAGA     3060     - CCGGGTGACA GGTTTTGTTC ATTTGAGAAG GCTTTCTTGA AAAGGGCAGT GA - #GCAAGCAG     3120     - GTTGGGAGGA AGAGGTGTGG CATTGAGAAG AAGGGAAAGT ATTGCATGAA AA - #AGTAATTC     3180     - TTCACGTGGA ACAGCCAGTA AGGAGGGGCA TGAGTAATAT AGGGTCAGCA GT - #TACTGGAG     3240     - CCAGAATACA GACTTTGGCC TGGGGAGTTC AAGAACTAAG AGTGGTAATA GA - #GAGTTGGA     3300     - TATTCCATTT CCCTTCTCTT TTTGTGCCAC CACCCAAAGC TCTGCATAAT CT - #AAGAAGTT     3360     - CCCTTGTTGA CACATAGCTC ATACTTGTGA AGTTGTACAA CAGGATAGCA TA - #GTGGCCAG     3420     - AAGCATGGAC AGTTGAACTC AGATATGCTT GGGTTTGAAT CTTACCATCA CC - #ATTTACTA     3480     - GTTCTGTAAT ACAGTGCAAG TTACAGACAT CTCTGCACCT CAGTTTTAGT AT - #GTCTAAAT     3540     - TGGGGATGAT AATGCCTTCC TTGTGGGGAT AGTGTGAGGA TTGAATAAGA TG - #AATACACA     3600     - TGGCTGAGCA CACAGCAAGC ACTAAATAAG TGCCAGTTTT AATGATAACG GT - #GATGATGA     3660     - TGATGATGAT GATGATGACG TAACATTGCT TGTGGGACTC CATACAGCTC AG - #TAGATGCT     3720     - TGCTCAAAGA AGCAAGTTAC CAAAATTTTT GTAATGGTTC TATGAACGTG AA - #AAAAGCAG     3780     - TCAACTTCTC TGAGGATCAA TTTCCTTAGT TTCCAATTAG GAAAAGTCTT CT - #TAGCTCCA     3840     - GAGTCCCACA GGGCTAATGG AATAAGGAGA GGATAGATCA CACATGTATT AT - #GCAAACAC     3900     - AACTCAGGTG AGCTCTATTC TTCCTTCTCA GTTATCCCTT CTGTAGGGAC CC - #CAGTGTCC     3960     - CCTGCTGTCT TTCTGTGTCC TGACCGGGAA ACACAGTGTG CCTTGTCTAC TC - #CATCACTT     4020     - GGCCAGCTGC ATGCTTTCCT TTGCAGGCTT GAAGCAAAGC TGGGTCTCGG AC - #ATTCTCAG     4080     - GCACTGACAA AGCTGTTTAG TTGTTGCTGG GAAACACTGG GAAATAGCCC TT - #TTGTTAAA     4140     - CACACAGAAA CTAGCCTTCG CCCTGAGCCA AATTCCTTAA ACTCGTCTAT GA - #AATTCCAT     4200     - AACCTGACTC CTTAACTGCA GACATACCCA GCTAGAACAT CCCTCATGTC CC - #TGTCCACC     4260     - GTGAGAATGC TGCACTTCAC TCTGAACCTT TAGTCCTCCT TTTAAATACT GC - #ACACTGAT     4320     - CACCCTGGTG TTTAGTGCTT TGTTTTTTGG AATCCCACCT GGCTCCATTT TG - #GGATGGTT     4380     - CCGGGCACTT CCCTATGGAA ATTCCCCTGC TGTCACTGTC AGAGTGAGTC CA - #GCAGTGGG     4440     - TTTAGCTGGA TGAAACACCA CCATGTCCAT TTCCATTCAG ACTAATGTCA GA - #ATTTGAAA     4500     - GGCACTATGG TAGAGTAGAA AGAACAAGGA ACTGTACTAT TTAAAGGGCA GG - #CAAAGAAA     4560     - AGGCATCTAT AGCTTATAAG ATGTGTGGAT CTTTGGATGT GACTTGGCCA TC - #CTGAGCCT     4620     - AAGTTGTCTT GTAGGAGAAA TGGGAATGAG AATATTTTCC TCTAGACATC AA - #GAGGAAAA     4680     - GAAATATAAC GTGAAAACCT TTGTGAATTG TGAATGTGTT ATACAGAGTA GC - #TAAAAGAA     4740     - TTAAAAAGGG AGTGACAAAA AAGTAAAAGG CAGCTGGCTG CTCAGGGCCT CC - #ATGGAGGG     4800     - AAGTACCTTG ATATGGTCAC TGTGGCTCAG TGACAGCTCT GCAGGGACAG GA - #AATTGATT     4860     - TGTTAGTGCA CCCAAAGTTG AATCTGCTCC TGAGTACTGA TTTATGGGAA CC - #AAACACAC     4920     - AAGAGATGAA GGATGTGTCA ACCAGAATGT CCAGCATTAG CTTGTGGGGA AA - #CACATACT     4980     - TCCAGTGACT GAAATACCAT CCTGTTATCA AGAGATCTGG GAAACTAAAG TA - #CTGACAAG     5040     - AGCTGGCTTG ATCTGTGGAT TTAGAACAAT GAGAGTTAGG TGGCCTTGAG GG - #AGATGATT     5100     - CACTCTCCTT CACAGAAGAG CTGACCTCTG GGGTCAACAG ATATAGCACC TC - #TTTCCCAG     5160     - GGACGCTACT GAATGAACAG TGATGTGTTC TTATACTCTG GCCCAGATTT TC - #TACATACT     5220     - TTCTTAGGTT ACAACTTTAT TTAGTCACAT TTCAGTACTG GGGATACTCC TG - #TTTATCTT     5280     - CTTTGGACTC GAGTTTTTAT GGGAAGGTCA TGAAACAGAG AAAAATACAA TT - #TGCAGGGA     5340     - AACTTACCAA GGCTTGTAAG GTTACAAGGA TTAAATGAAA ACCCTGTGTA AG - #TCAGTATA     5400     - TAGTGAAGAA GTAAATTGAG TTAGACCAAA CGCCAAAATG CATCCGCATT AG - #AAAGACGA     5460     - TAAAGGAAGA CTCTGGATTC AGTTCTGTTC AAAAAACATT TTCTGCACAA AT - #ACTATGTA     5520     - TGAGGAACTG GGCGTTGGGG AGATGATGAT GAGTGAGACA TGGTTCTTGC TT - #TCAGAGAG     5580     - CCTAGAGACC TGGGTGGTAG CAATGGTAGA GATACATCCA AGACACAGAA AT - #AGATATAC     5640     - AGGAACACAG ATGATTGAAA GTGATGCTTG GCAGGGCTTT AAAGAATGAA TC - #AGAGTTTT     5700     - TCAGGCAGAC GAGGATCTTC AAGGCAGAGG GAATCATATA GATAAGGACA TA - #GAAGAGTG     5760     - AAATTTCATG AAGTAGTTAA GCATCTGAAG AAGCATGGAA TTAGTGACAA GA - #AATGATGC     5820     - GGAAAAGATA TCCAGATCCA ATCAAGAAGG GCCTTGTTGG CATTCTATGG AG - #TCTGGACT     5880     - TTGGCTTCTG GGTCACAAGT TCTCAGATGG GGTTTTCATA TCTATTATTA GA - #CCTACTAT     5940     - GTACTGGTCC AGTGGAAGGG AAAGGGGTTG TCTTACTGCT AGTGGAGTAG GA - #ATTGGGTA     6000     - TGGACCACAG CTTGTCTTGT TTCCAAGTAT TCCCTAAGAA ATCTGGTCTG CT - #GATGGGAG     6060     - ATCTATTTAT GGAAATGTCT TTTTCCCTCA GGAATTTTAT GTCGGAAACA GC - #TGTCATAG     6120     - GTGAGGAGGA ACTGGTAAAA GTACTTAATA GGAGAGTGTC ATGGTCAGAT TG - #GTGTTTTG     6180     - GAAAAGTCAG CCAGGGCAGA TTGGAGAGGT CCATATTGGA GGCAGGAAGA CT - #TAAGAGAC     6240     - TATTGCAAAG GTGAAGACAA AAGACGATAG GGACTTGCAC TTTAATTCCA GC - #CCTTAGAA     6300     - GTAGTAGAAG GTCAGAAATG AGAATATGCA TTACAGAGAT AGTTAGTTGC TA - #TATCATTA     6360     - GGACTTGGTG ATAGATTGGA TGAGGATGCG GTTGGGTGAG GCAAAGAGGA GA - #GTCCACAT     6420     - TCCTGGTCTG GGTAGTAACA AAGAATCTAG CAAGAGGGCT TGTGGGGAAA GA - #TGCTGAGT     6480     - TACGTAGCAA GTGCATCTGC TTTATCCTTG TAATGAATGG GGCTAAAGGT GT - #AAACCAAA     6540     - GAGTCATCAG CATTTGGAGG GTAGAATAAA TCATCAGATA ACTCAGGAAG AA - #GGAGCAGA     6600     - AGAATTACTG ATACTCCCTG GAAGGAAAAC CGGAAGTAAA TGGGAGAAAC TT - #GCTCAAGT     6660     #      6693        ACAT GAAGCATGAA TTC     - (2) INFORMATION FOR SEQ ID NO:  3:     -      (i) SEQUENCE CHARACTERISTICS:     #            24979 base pairs     #              nucleic acid     #        singleTRANDEDNESS:     #           linearLOGY:     #       DNA (genomic)YPE:     -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  3:     - GAATTCCAAG CTTTCTGCTG TAAGGAGGGA CCTCAGGGAG CCAAGGTCAG CC - #TGCAGCCT      60     - TTCTGTGCTC CTTTGCCTCG CCTGACAGGT ATGAGGATGA AATCAACAAG AG - #GACTGGCA     120     - GCGAGAATGA CTTTGTCGTC CTGAAGAAGG TGAGGGAAAG GGGAGTCCTG AG - #GGTGGCTG     180     - TGGACCCAGG AGGCTGAGGG GAGTGAGGAA TCCCTATGGA TGCTCTGTGA CA - #ATGGCAGG     240     - GTGGCCTCTA CGGCCGGCTT GCTGTGTATG ATGCCTGAAT GCGGGGCCCT TA - #CATTGGAA     300     - CTGACACTGA TAATGACTCT TCAGGAAGCC TTGAGTTCGT ATCTCTCTGG GG - #TCTGAAAG     360     - TGAAATGAAG TGAAATGACA GCTTTTGAGT GTCAGTTACC TGTAGCCTTG GG - #ACCTAAGG     420     - AAGGACCTGG GGTGTTGGTT GTGACTGACT GGGATGTGGA GGTTGGTGTC AC - #ATCTCCTT     480     - CTGGCCAGGA AAGCCAGGAC TTGTGGGTCC TTATTCGAGT GCGGTGATGA AT - #TTTTTAAG     540     - TAAGGAAATA AACCTAGAGT GGCTCTGGTC CTGAGCCAGC CAGTGAGCTG TG - #GCAGGCAA     600     - TGCCTGGGCA ATAAAGTCAA ACTGTTCTGC CTGCTATTCA GGATGTGGAT GC - #TGCTTATG     660     - TGAGCAAAGT GGACCTGGAG TCCAGGGTGG ACACTCTGAC TGGGGAGGTC AA - #TTTCTTGA     720     - AATATTTATT TTTGACGGTG AGTTAAGCCT TTATAAGAAC CTCCTTTCTT TT - #CTCACATC     780     - TCACAAGGAG TATGGGCTGT AAGAGGGGAG GCCTGAAACC CAACACTACC CA - #CTAGGGAC     840     - TCATCTCCCC AGGTACCCCA ACTCTGTGGG CCTGGAGTCA GCCATCCTCT CC - #ACCCCAAT     900     - CCTCAGAATC CCCAGGTCGG GGTAATGAAG ATGGAAGGCT GGGAGAATCC TG - #AGTTAGGT     960     - GGAGGCGAAT GTGTCCCTGG TTCATGGCTT CCAATCTGTC TGGGAAATCA CC - #CAGACATA     1020     - TAAGGGGCAA AACCAACCAG AAATCTTCAT TAATTCTGGG GAGTTGATGG AG - #CTGTTAGG     1080     - AACTCTGTGG GAGGTGACAG TGTGAGTCTC AAGGAGTGGA CTGACCTTAG TG - #ATGGGGGA     114     - TCAAACACTC CACCACCCGG CCCTCTTTTG CCTGTGTCTA ACTTGGGGGT AC - #GTGCTCTG     1200     - GGCCAGATGC TGTGTTAGAA GTTTATGTTA TGGGTATCTC CATTCTACAG AT - #GGGAAAAC     1260     - TGAGGCACTG AGGGGTTAAA TTACTTGCTT CATTACCTAG CTAGTCAATG GT - #ACAGCCAA     1320     - GACTCAAAAG TGAGTCCAAG TGACTCCTTA ACTAGAGTCC ATCTACTGCC TC - #GGAGTACT     1380     - CATGTGGTTT CAAGGAAGAG GCATGCCTGC CAAGGAGCCC AGCTCACTAT GG - #TGGCCAAG     1440     - TCAGAGCAAG GCAGAGTGGC AGCTGCAGGA GAAGTGTGAT GGGGAGATGG TA - #TCTGAACG     1500     - CTCCAGGTTT AGGCTCCTTC CTTCTCCCCT GGAAGGCAGT TAAGACTCTC CC - #TATTATCT     1560     - CTCATTGCAC ACAACAATTC CAAGAGCTTT TCCCAAGACT ACCTGGCCCA GG - #CTTCTGGC     1620     - TTCCCCCGAG AGCCTTGAGG GAGCAGCAGA GGAAAACTGA GGCCCCCAGA GG - #AGAATGGA     1680     - AGGAGTCAGC CTGTGCGCCA TGCCTCGCAG GAGCTGTCTC AGGTGCAGAC TC - #ACATCAGC     1740     - GACACCAACG TCATCCTGTT CATGGACAAT AACCGTTCCC TGGACCTGGA CA - #GCATCATC     1800     - GATCGAGTGC GGACCCAGTA TGAACTGATT GCACAGAGGA GCAAGGACGA GG - #CCGAAAGC     1860     - CTGTACCAGA CCAAGGTGGG CGTGGCCCAG ATCTGGTGCC CAGAAAAACA GA - #TTCTTCCC     1920     - AGAATTGTCT TTCTCTTATT GCATTGTCTT TCTCTTATTT CTGAAGTAAA AT - #GTGTTTGT     1980     - TATACAAATT CTAGAAATTA CATGTAAAGA TTACCCATCT CTCACTACCG CT - #ATTAATAT     2040     - GTTAATATCT CTTCTACCAG TTCTTTGTCG CTATTAGGCT AGTGGAAAAG TG - #ATTGCGGT     2100     - TCTCGCCATT AAAAGTAATG ACGAGAACTG CAATTACTTT TGCAAAAACC CA - #ATAATGTT     2160     - TATTGAGAAC TCATATGTGT TAGGCACCTA GCAAAGTGCT TTACTTATTT AT - #TATTATTT     2220     - CATTCAGTCC TTACAACAAC CAATGAGGTA AGAATTTCGT TATCACCATT TT - #ATAATAGA     2280     - TAGTAGTGTG TGACATTACT TAATTTCCCT AATGCCTTGT AGCTAGTAAA TG - #CAGAGCCA     2340     - GAGCTTAATT AAAATTGGTT TGTGTCTACA AACCCATTCC CCTCACCACT AG - #AATGATTT     2400     - TTATTCTTTT TTCATAATGG TATCTATTAA AATATATTTT TTTTACTTTT TT - #TTTTTTTT     2460     - GAGATGGAAA CTCACTCTAT CTCCCAGGCT GGAGTGCAGT GGCCTGATAT CA - #GTTCACTA     2520     - CAAACTCCGC CTCCTGGGTC CAAGTGATTC TCCTGCCTCA GCCTCCTGAG TA - #GCTGGGAT     2580     - TACAGGCACG TGCACTACAC CAAGCTAATT TTTGTATTTT TAGTAGTGGC AG - #GGTTTTGC     2640     - CATGTTGGCC AGGCTATACT TTCTCTTTTA CTTAACACAT ATGGATACTT TT - #CTGTGACA     2700     - CTAAATAACC TGCCGCATTT TTAACAGCCT GGTATTATAT TGTTAGACTA CA - #CCCTCCCT     2760     - TATTAGATCA ACTCCTTTGT GGCTAAGTTG TTGGGCACAT CTTTGGTTAC TT - #CTTTTCCA     2820     - TAAACTGACC TGGATCTTTT TGGATGGTGA AGCCTCTGGT TGAAAGGGTG TG - #GCTGACAG     2880     - TCCAGTCACT AAATTCTGAA CAACTAGCAT TGATGACTGG CTTTGAGGAT GA - #TCTGTGGC     2940     - CAACTCCAAT CCTGGCTGAC CTCTGTCCCA CGGTCCTGCA CAGTGTTCTG GG - #GTGGAATG     3000     - GATTTCGACA TTAGACTAGG AAGCCAGATG GCCAACAGTG AAAAATAGCA GA - #GTGTACCA     3060     - GATTCCCTTG CAAGTCGATG CTTCTCCTAC CCACTTCAGA GCCCTGTGCC TG - #GGGGGTGG     3120     - AGTTCTGACT AATGGGGCAA TACAGAGACA GAAACAGAGA TGGAGGGGAA AT - #GAGACTGA     3180     - ACGTGGAGCC AATGGAGGGC CTCTGAGGAC ATGAGGTCTG CTTGACTGCT AG - #GGAGATCA     3240     - TCCTGGAAAA GGGTGGGAAG CTATATGGTG GGTGGAAAGA GTGAGGGGGT CT - #CAGTGTGG     3300     - GTAAGGACCA ACGTGAAGGC TTAGATGTGT GAAAAGGTGG TAGAAGGGCA TC - #ACAAAGCA     3360     - GGTTTGTCTG GCCTGGGATG AGAGTCTGCC CAGAGACTGG TGGGAATGCG GG - #AGGCTTGG     3420     - GATAGTGTGA GTGTGTGCAT GTATACATGT GTTTGCAGCC TGGGTGAGGG AG - #GTTTGGTA     3480     - TAGCTGTGAG TATGCATGTA GGGGTGACCA CAGTGCAAGG TGGGTGGGAA TC - #TCCCAGGG     3540     - GAGAGCAGCC CAGACCTACT CCTCCTGGAG GGGCTTGTGG TGGGCAGCAC AT - #GCTGACTA     3600     - TGATGCTCGC TTTGGCCCCA GTACCAGGAG CTCCAGATCA CGGCAGGGAG AC - #ATGGAGAT     3660     - GACCTGAAGA ACAGCAAGAT GGAGATTGCA GAGCTCAACC GCACCGTCCA GA - #GGCTGCAG     3720     - GCAGAGATCA GCAACGTGAA GAAGCTGGTG GGACGGGTGC TTAGGGAGGG CT - #GACCAAAG     3780     - CCCTGCACCT CCTACAATGC CCTGCCAGAT CGAGCTCTGG AAACTTAACC AT - #TAAATGGT     3840     - CTCCAACTGT CTCTGGAGCA GATTGAACAG ATGCAGTCAC TCATTTCGGA TG - #CTGAGGAG     3900     - AGAGGCGAGC AGGCCCTCCA GGATGCGTGG CAGAAGCTGC AGGACCTGGA GG - #AGGCCCTG     3960     - CAGCAGTCCA AGGAGGAGCT GGCCCGGCTG CTGCGTGACT ACCAGGCCAT GC - #TGGGGGTC     4020     - AAGCTGTCCC TGGATGTGGA GATCGCCACC TACCGCCAGC TGCTGGAGGG CG - #AGGAGAGC     4080     - AGGTGGGTCT GGCAGCTGTG TTTCTGGGGC TAAGGCTTGA GATGCACCAT GA - #AGCTGTGG     4140     - GACTGGCTAT TTGGAGAAAA GATAAGCCCA CCTTTTTGGG AAGATTGGTA GC - #CAGGTGAG     4200     - CAGAAACATT CCAATTAGAG GCAGAGGCTG TGTGAATGGA CAAGCCTCTT CA - #CACAGGGA     4260     - GAAGTCATTG TTATCATTCC TCCACCTCCA AGTAGAATGT CCTTATACCC CA - #ATCCAAGC     4320     - CTCTGCAGCT GGTATTCACC CCCAATGCTA AAAGGCTTCA TGAAAACCCT GA - #AATTTCTC     4380     - TCTGCCCCAC TGGCTTCCTG ACCTCTGCTC ATGCACACAC ATTTCCCTAA GG - #CTTGGGGA     4440     - CACCTCTGAT CCAGATGTCT GTGGCCACAG CCTTCTCTCC TCAGGCCCTG TG - #GGTCTGGC     4500     - TGACCGTGTG CTTTGGTTTT ACAGGATGTC AGGAGAGCTG CAGAGCCATG TG - #AGCATCTG     4560     - TAAGTAGCAG ACCCAGGGGC AGAGAGAGGC TGGTGGTGCT GGGTGGAGGG AG - #GGCCAGGA     4620     - GGTGGCCAGC AGAGAACGGA AAGTCTGGCA TTTTAGCTTC CAGTCCTGTG CA - #ATAGACAC     4680     - CAAAGTAAGC AAGTGTAATG CAAAGCCTGG AAGAATTCAT TTCAAATAAA TG - #GTTATGAT     4740     - TTCAGGTCTG CTTATCTTAA TTGTTATGAT GCCTTTTTAT TAAATGATGC CT - #AGGAGGAA     4800     - TCAGCAGCGG CTAGAACTCT TTAGGGTACA TATTCAATAA ACAATGTAAG TG - #TGTTGCTG     4860     - AGAGGAACCC TGGCATCCCT TTGTAGTATG AAGAATACTT TTCAAGTAGG AA - #CACTTTCA     4920     - ATTTTCAATG TATCGGGTTT GCAAGTCGAT GCCACGGGTG ATCGAGGATG GA - #GGAGGCTG     4980     - CAGGTGCAGG GCGGGTGCAG GTGGGGGCCT TGCCTGTCCC TCTGACCCCG TG - #TGCACTGT     5040     - CCCTACCCAC AGCCGTGCAG AACAGCCAGG TGAGCCGTCA ACGGCGGCGC GG - #GAGGCGGC     5100     - GGCAGCTACG GCTCAGGAGG CTACGGCGGC GGCAGCGGTG GGGGCTATGG CG - #GCGGAAGA     5160     - AGCTACCGCG GAGGCGGGGC ACGAGGCGGG AGTGGAGGCG GTTATGGCAG CG - #GCTGCGGC     5220     - GGCGGTGGCG GGAGCTACGG AGGGAGCGGC AGAAGCGGCC GCGGATCCTC GC - #GCGTGCAG     5280     - ATCATCCAGA CCTCCACCAA CACCTCCCAC AGGCGGATCT TGGAGTAGAG GC - #CTCGTTTC     5340     - TGCCACACAT CACGCCTGCC CCTCACCGAC CTCTCCTCAA ACTCCTCCCC TC - #CACGCCCT     5400     - TCCTAATCCC CTCTCATTCA CTTTTCTTAA TGGGTCTCAG CAATTTTGCC AA - #TAAATTCG     5460     - ACTCTAATGG GGGAAGCAGG GTGGATAAGT CCAAACAGCA GATCTCTCTT TT - #GGAGGGCA     5520     - CTGGCTTGCA GTCAGATTCA CAGCTAGGCA CATTCTCACT CAGACCCCGC TC - #TGCTGGCC     5580     - CTGCTGCTGT TCCTGCTCCC ACCTTTTTGG AAGATCGGTA GCCCAGGGTG AG - #CACAAACA     5640     - TTCCAGTTAG AGGCAGAGGC TGCGTGAGTT GGCAAGGTAG GGAGAAGTCA TT - #GTTATCAT     5700     - TCCTCTGCCT CCAAGTAGAA TGCCCTTATG CCCCAGTTCA AGCCACTGCA GC - #TAAGTATT     5760     - AACCCCCAGT GCTAAAAGAT ACCAGGCATC TAGTTTAGCA ATGGAGGGAA AC - #AGAAACAG     5820     - CCTGTAGAGA GCATCGACAA GGCGCATAAT GGAGAGTTGG TATCTCAACC CC - #AAGCTCCC     5880     - TTTGCTGTAC CTGGGCCTGC TCTGTGAACA AGAATCCACG CCCCCCTGCC CT - #GCTGGGAC     5940     - CACATAATGA TCCCTTTGGG GAAGTTGCTG ATTGCAGGGC AAGCTAGCTT GG - #TAAGGAAA     6000     - ACCTCTGCAC CAGCGGCCTA TTCCTGCCTC TTGGTCCATA GCCTCATACA CT - #CATGTTGA     6060     - TGGATAGTAT AGATTTGCTG CCCACACCAG ATATCTGTAA GGCATCACTG TC - #CTGATTCT     6120     - GAACCTCTGT TTCAGGAAGC ATTCTCCCCT GTGTAAACAA CTCAAGGTGG AA - #GTATTTCA     6180     - GAGGGCATAG GGTCATGAAT CCTTACCCAA AGGAAGCCTG TTTTAGCAGT GG - #ATGCAGGA     6240     - GTTGATGAAC AGACAAGCAA GTTCTGCTTC TGTCCTGTTT CCTCCTGACA GC - #TCCATTCT     6300     - TTTGAAGCCT GACCCTTCCT AAGCTCTGCA TCATAACCAC TCTGAGAATT GC - #CCCATTGG     6360     - TGGGCATGTG AAGCCAGCTC TGTTCCATCC AGGTGCCTCG GGCCTGAGGA GT - #CTGAGGAT     6420     - CTGACTTGGG TCTTGGAAGG GTTCCAACCC AAGTCAGTCA GGAAGCTGCC CA - #TTTTTTTG     6480     - CAAGGCATTT TAATGCCTTT CCCAGACCTC TCTAGTCCCT CCTGCCTTCT GT - #TCTCTCGA     6540     - CAGCTGTGAG CCCTTTAGAG AGAATATGAC TCTTAATTTT GAATCTTATG TA - #AGAGGCTT     6600     - GAGATGTGCT GGAGAGGGCA GGAAGAGGAA AGTATCAGGC CTTGAGAGAG GG - #AATGTAGC     6660     - TTTGCTTCTA TGATCTGGAG TCACCTTCAC TTGCTAGCTG AGTCCTAACA CA - #ACTTCCAA     6720     - GTCCATGATT CTCTTGGGGC ATTGGATGGG CTCAGTGTGG GTCTCTTAGG CT - #GTTCTTGT     6780     - GACTTCATCA TTTCCTGGTT CAAAGTTGTA CTGTCAAGGG GCAGCATTTC TG - #GTATTTCT     6840     - ATAATAAATT TTCTGTGATC TCAAATTGCT GTTTGGTCAG GAGATGCATT AT - #TTCTTCTT     6900     - CTTCTTCTCC TTCTTCTCCT TTGCCTTCCT CCTCTTCTCC CTCCTCTGCT TC - #TCCTTGCT     6960     - CCTCTGGCTC CTCCTCCGCG CTCCTCCTCC TCCTCCTCCG CGCTCCTCCT CC - #GCGCGCTC     7020     - CTCCTCCTTC TTCTTCTTCT CCTTCTCCTT CTCATTCTTC CATCTTCATC TT - #CATCTTCT     7080     - CCTCTTTTTC TTCTTTCTTC TAAATAAAGA TGGGGTCTCA CCATGTTTCC AA - #GGCTGGTC     7140     - TTGAACTCCT GGGCTCAGGT GATCCTCCCA CCTTGGCTTC CCACATTGCT GG - #GATTACAG     7200     - GTGTGAGGTG TGGTACCTGG GCTATTTCTT TAAAAATTTC TGCAGACCTC TG - #AAATTATT     7260     - TATATTTGGG AAGTTAAAAT TTCTTCTTAT TTTTTATTGT ACAAGTAATA CA - #CAGTCTTG     7320     - AAGAATCTTA CAGACATAAT CTTATTAATC CTTAAAGTGG CTGATCATCC AA - #AAGTCAAT     7380     - TATACATTTG TTCAATGAGC ACTTATTAAG CTCCTACTGT GTGGCGGGCA GT - #GGCTTAGG     7440     - CACTGGGAAT GCAATGTTGA ATGAACATGT TTCTGACTCT TAAGTTGCTC AC - #AACTAAAT     7500     - GACATATTAT GGGGGAGGGA CGATTCAAGG AGAGAAGAGA AATCTGAGTG TG - #CTTCTAAG     7560     - GACCTCTAGC CTGAGAGTGG AAGCAAGGCC TATCCTGAGG ACACAGGCAG AC - #CCCCCAAA     7620     - ACAGGAACAG GTGGGACTTA CGACAGGTGC CAGTGCTGGG GAAGGGACGT TT - #GGTTCCAA     7680     - CAGACTCCTG GAGGACTGGG ATATGGAACA GGGCCAAGGA AGAGAGGTGT GG - #GTGGGGAG     7740     - ATGAGGGAAG GGCCCTCCAA ACAGGGGGAT AGTCTGCTCA GAGACTCAAA AT - #AAGAGAGA     7800     - GTGTGGGGGT GAGAAGGAGC AGCTGGACAG GAGAAACTGA GCTAGGGAAG GA - #AGGGGCTG     7860     - AGGCCACAAA CTGAGTGGGG TCATGGGCAG AGACATCTTC AATTGATGCC TT - #GAGGGAAG     7920     - CAGAGATGCA GAAATTCCAT AATGGAGCAA GTTAAGCCAT CACCTCATCC TA - #TGTGGTAG     7980     - TTCTCAGTCC ATGTAAAAGA ATCACATAAA AGATGTGATC TACTTTCTAA TT - #CCCTGGAG     8040     - GACTTTGCAT GCAAATTTGG ATATGGGATT CATTCGAATA TGACAGGAAC CC - #CATATTGA     8100     - TAAGACACTG TTGCTCCCGG GTGGGCATTG TTCAACTCAA GACTTGATGA CC - #CAGATAGG     8160     - TGTGTCTTTG CAGTTAGCTG TCACATGTCC CACCGTTGAA AGGTGGGCTT CT - #CCTCCACA     8220     - TGTGCAGGGC TCTCTGCCTG CCTTTCCCTT TTCTCGTGTC CTCTGACAGC CT - #GCTGCCAG     8280     - GATAGATGAG ATGGGGAGAA ACTTCTCAGA GAGAATAGAG GGGTGTGCAT GG - #AAACAGAG     8340     - TGTCTTATCA CTATGGGTTG ATATGATGTT TGCAGTTAGC TGCCACATCC TC - #CCCAAAGA     8400     - CTTCTGGAGG GCATGCCTGG GAACACAATG TTTTATTCAT ATGGGTTGCT GT - #CCTATTCC     8460     - AATGAATCCC ATATCCAAAT TCCATCAATA TCGCCTTCAG GAAGCTACAA CA - #TATTCGGC     8520     - TCAATATAAG AAGCACCTTT CTATGATCCT GACATGGGAG AGGCTACCCT GG - #GGAGTGAT     8580     - CAAGTTTCAA GTCAGAGATT GGCTAACCGT TTGGCAGGAA CGTTGAGGGC GG - #GAGTGGAG     8640     - ATGGGTGGGG ATATGGTATG GAGGCATCTC ACTACTTTGC TGTACTAAGA GT - #TCACATGG     8700     - CGAAACCTGA GAAAAAAAAT TCTACTCTCT GTGTTATATG GGAAGAATAA GG - #TCAGGTGC     8760     - CAGTGAAAGC TAAAGTCACA AAGAAGCCAA AGGCCCTAGC CAGAACTGTT AA - #ATGAGGCT     8820     - AAGTTTTCTG GCAGCACAGG GTCTATTACA GGGTGTGAGT TTGATTATCC CT - #GGGATCAT     8880     - GCATGTGTGA TACTCTAATG GGATCCACGT TGGCTCTGAG AAAACACGCA AG - #GATAAGGC     8940     - CAACCACAGC TCTCCTTTCC CATCCTCTCT TGGGAACAAG TTGAGATTGT CC - #CAGAAAAT     9000     - GTGGCCCTGA CTTATCTCTT CCGAATTCCT TGATTTTGTC CTGTCATGGA GG - #CCTGGGGG     9060     - ACAGATGGAG GGAATCATGT GCCTGAATCT GAAGAATATT GGAATAGAGA TT - #CCACAAGG     9120     - TAGGGGCAGG AGAAATAAAG GACAGAAAGG AGAGGAGTTG GTCAAAGAAG GC - #ATCTCAAC     9180     - GTCTAAATGA GAAGTCTTAA TTCGATGTTC AGGGAAAGAA AGAGTAACTT TA - #GGGACCTA     9240     - AACAAGGAGG ACTAGCACTA AGACACTGAA GAGATTTCCT GAAATAGACA AT - #ATTTCCAT     9300     - CAGAGACAAT GAGAAATCCC ATCAGGAGAA AATGTCTCTC ACTTTCAGCT CA - #CCCCAGTG     9360     - AAAACAACAA GCATTCTATA AACCATGTAG GAAATGCCCA CACATGCATT AT - #CTCACCTG     9420     - AGTCCCACTC ACCTGGGAGT GCGGAGACCA GGTGTGGGGG TCTGCAGTCC TT - #CTAGGGAC     9480     - CATGGAGTGC TCCATCCCTG CCCCTAATCA ATGCTATTCC CACAAGGCAG AT - #ACTCAGAG     9540     - GGAGAGCCAA GCAGGCTCAT TGCAGTGCAA TAAAGCCAAG AGGCTGGCAG GA - #GGGAGCAA     9600     - ACACCCGGGT TGGTGAGAGT CCCAGGGAAA GTCTGCCAGT CTGCTCTTTG CT - #CTGAGAGG     9660     - CAGGGTGGCA GGGTTGGGGC ACTCTGGAAA TATAAATTTA GTTCCACCAG CT - #TCTCATCC     9720     - ACAGAGATTT TGATCTGAGG ACATGGTTAA CTGGAGGAGC AATCATTGAC TC - #AGTAAAAT     9780     - TCTAACTGCA TCTGACCTTA GACAAGGTGT GCGTTTCTGG GCTGGGAAAG TT - #CCTGGTCT     9840     - GAGGAAGAGT CTCTTGAGAA TGTCATCTCT TTTCAATTAC CCAGCCTTTT GG - #CCCAGAAT     9900     - GCATCTTCAA ATTAATGAGC CATTTGCTGG TTAATTTGGT CCCAGGGAAA AA - #AGTCCAGC     9960     - AAATTACTGG GCATTACACT GAGCTTGAAG GTCCCTCTTC AAGGTTGCCC TG - #GTTTTATC     10020     - AGCTTTCCGA TCAGTCTGGG AAATGGGATG TCTTCAAGGC TGATCAATGC TC - #TGTTGAAG     10080     - GGCTGGCTGG GAATTTGGGG TATTGGGAGG TTTTCTAGCA TGGAGTACGG CT - #CCGAGTGG     10140     - CCCAATCCCA AGCCTGGAAG GGCTTCCAGG GGGCTCTAAG TGTGCATTCT GA - #CCTCCACA     10200     - CCTGCCCCTG TGTGCTCAGC CCTCAGTGTT TGTGCTCCCC CTGCAGAGCA GC - #TCTGCAGT     10260     - GAGGGCAAAG GCTCCTCGCA TCTGGCCCCA GCTCCCTCCA GCCTCAGGTG AG - #CCCGGTGA     10320     - TGCACCTGTG ATCTCTTCTT CCATGTGATG CCCCCTAGCT TTCCCAGGGC AA - #GTCCGTGG     10380     - ACTTCTTAAG GCTTTCTCTC ACAAGATGAG GAAATGGGCC CATGTCAAGG GC - #TTAAATGT     10440     - CCTGTTCCAG CCTTTTCACT GTTTCCAGTA AATCAGGGGC TTGTTCTAAA GT - #TTGTTTTT     10500     - TTTTTTCTGG TTATTATATC AGCTTCTGGG TTCTCTCAAA TGCAAGAGTG AG - #GGAAAATC     10560     - TTCCTTTTTT CCTTTTTTGA GATGGAGTCT TCAGCATCAG TAGCCCAGGC TG - #GAGTTCAG     10620     - TGGCGAGATC TCGGCTCACT GCAAGCTCCG TCTCCTGGGT TCACGCCATT CT - #CCTGCCTC     10680     - AGCCTCCCGA GTAGCTGGGA CTACAGGGCC TGCCACCACA CTGGGCTAAT TT - #TTTTTGTA     10740     - TTTTTAGTGG AGACGGGGTT TCACCTTGTT AGCCAGGATG GTCTCGATCT CC - #TGGTCTCG     10800     - TGATCCGCCC ACCTCTACCT CCCAAAGTGC TGGGATTAAC GTGAGCCACC AC - #GCCTGGC     10860     - CCTTTTTTCT TTATCTATAC TCTACTATGC TTCAGTTTCC CTGGAAGGTA CA - #TAGAGCCT     10920     - CCTTTTACAG AGAGAACTAG CTCAGAGAGG TCAGTGACCT GCCTAGAGCA GT - #GCAGAATC     10980     - AGGAGCGGAG CCCAGCCTGG CAGCCTCCAT GGCACAGAGC AAGATGGGCC CC - #ACCGCCTC     11040     - TCTCCTCCAT GTTCATCTTT GGTTTCCTCC TTCCTGGCCT CTGCTCTGCT CC - #AGCCTTGC     11100     - TAGTGAGTGA CTCCTGAGGA CCTCCTTCTT TGCTGTCCAT CCTAAATAGG GC - #TGCCCCTC     11160     - TGTCTGCAGC TCTCCCTCCT GCATAAGAAG CCTTGCGCCC TCCTCTGCTG CC - #TGGCTGCT     11220     - TTCAACATCT CGCCCCGCCT CCCCATTGTC TGTGATTTCT CTTCACTCCA CC - #CAGGCCTC     11280     - AATTTTCCTC ACTCCCCTGG GATTTCCCTG TCCCATGTCC CTGGTGGAGT CC - #CTCAGGGT     11340     - GGGTGGTTGT CATGCAGTGC TTTCTTCACT CTTTTCTTGG TCCCATCCCA CA - #AAAGCTCT     11400     - CAAAACATCA CCACACCTGC TCCTGCCCAT GCCCCACAGC CACCCCTGGC AG - #CCTCATCT     11460     - CAATGATCAG TTCTGGGTTG TGTGTGTGAG TCCTTGGGTG GGGGTGTTTT GG - #TGCTCTGT     11520     - CATCAGCACC GCTGGGGTAA CTCTCAAGTA TAAGGGGCCA TGTGGGATGC TG - #GGAGGGCA     11580     - TCAAAAGACA CAGGGGACTT AGTCTTGCTT TCCAAAGGCT TCCAGAGTGA TT - #GAGGGGCC     11640     - AGGAAACACA CAAGCACATG CATGAAAATG AGCCAACAAA TGCATCAATA TG - #TACTAAGT     11700     - CTGGCAGCAG CCGAGCTTGG AAAAAGAGAC CAATAGAGCA CTTGCCCGAT GT - #GGACTGAG     11760     - CAAAACTCCC TGGAGGAGAT GAGATCTGGA CCTGTCTGCT GCCTGCTTTG AG - #TGAGAGAT     11820     - GAAGGCATTT GCCCACAAGC CCTGATGGAC CAAAAACAGA TTCAGGACCA AA - #TGCTCAGC     11880     - CATTGAGATC TTTGGTGCCC CAGAGCTTGA CTATGGGTAG GGATTTGTGG CA - #ATGCCGAG     11940     - GCAACCAGAA GACCTTTCAG AAAAGAGAAG AGTAGAAGTG GGCTTGGAAG AC - #AGAGAGGA     12000     - ACAGGGATGG AAAGGGAAGA AGAGGGTGAT CAGTCTTGGG CAAAGCACGA GA - #GCTGAAGG     12060     - GGTCAAGGCT GTGAGGCCGG GGAAGTGGGT GAGCAGGGTA AGATGTAGGT GG - #TGCTGGTG     12120     - GTGAGAGCAG GCCAATGACA GAAGGAGCCC ATGTGATGCG GCGGGCTTGG AC - #TCTGGAGT     12180     - GAGGCACGTG GCTTGTCAGT TACCTGCTGG ATGACTTTGG GCACATATTT CA - #ACCTCTAT     12240     - AAACCTAAGA TGCCTTTTCT TTAAAATGGG GCTAAGAGCT CCCACGACGC AG - #ACTTTGTG     12300     - TGGTATTTAA ATGCAATGTG GCTCCTAACA GCATAGTTGC TGCGTGTAGA TG - #TTAGTGTC     12360     - TCTTTCTTTC TCATTTTGTC TTTATTTCAT AAATGCACAG TCACTAAGTA AG - #AAAGGAGA     12420     - GAGTGTGTGG CTCACACTTT CCTGCATGTG GTTCTTCATA TCCCACACAC CA - #CACTGATC     12480     - CTGGGGACAT CACAGGAGAT GACGGGCCTG GTCTGGCAGC ACTGCAGCTC CA - #GCTCTGTT     12540     - GGGCTGCCTC GAAAGTGGGC AGTGGAAAAA GAAAAGGAGT TTGATTCAAC AA - #TTGGAAGA     12600     - GTCTCAGGAA TTGACTTATG ACTTGGACAC TTTTTTTTTT TTTTTTTTTT TT - #TTTTTTTT     12660     - TACTTTTTTG GGCCTGTGCT CTCACTTCTC TGTGAGGCAG GTTAGATGAT GT - #GACCTTTG     12720     - AGGCCCCATG GATGAGAACA TTCTGTAATT CTCTGTGTAC TTGTTTATAG GG - #CCCAGTTC     12780     - CACTTGCCTG TCTTTGAGCC TCTTCCCGGT TCAGGGAGGA ATGTCACTTG AA - #TTGAAATC     12840     - AGAAAACCCA GATTCTGCTT CCAGATGTGT CTTTTCCTAG CCGGAGTGTC TA - #GAGGAAGC     12900     - CACTTAATCT CTGAGAATCA GTTTTCTGTT TCATGAAATG GGTTGAGAAC AG - #CTTGATTG     12960     - CCTAGTTCTC AGGGCTCTTG TGGGATGCTC TTTGCATATG TGTTTGGTGG GG - #TGAGCTGT     13020     - GCAAATGTAA GCTATGGTGA GGTTTATGGC ACTTATTCCT GCTAGTCCTG CA - #TTTCTCCC     13080     - TTCTCACAGG AGCACCTGGG GTATGTTTTG CAGCTAAGTT GTCTACCAAT TC - #CCTGACCA     13140     - TTCATTCAAA CCTTTGATTT TTCTGTATGT CAGTTTCTTA GTTCAAAGAT GG - #GAGTGTGG     13200     - ATCACTGCCA AGGTCTGTTT TTGGCTGGCA CACACATGCA CACAAACATG TG - #TGCACACA     13260     - AACATGTGTG CCCAAACATA CTCACACCCC TCCAAAATGC TAGAAGGAAT CG - #ATTGTGCA     13320     - GAACAATATG TCTCATGAGG GAGTATGCTG AACTAAAATA ATTTTGATTG CT - #TGTCAGAA     13380     - AATGATTAGG CAACAGTCAT TACCATGCCA AGACTGTCCC AGTCTCCATT GT - #TCCTAACA     13440     - AGACCTGAAT TACTCATTCC CTAAAGAGAT GGTTGGTTTA GCAGCCGAAG GA - #TTTTAGTG     13500     - CTAGACAGAG TCCCAGACAG CAGTGCCACA GTGATGGCGA GGGAGAGGAG TA - #GCAGGGGA     13560     - GCGGTGAGGG GCACTTTCTG GAGGAGGGTA TAGGGCAAAA ACTGGGAGGA GA - #AGAGGGAC     13620     - AAGGTTCAAT AGCGGAGTGC AATGGAGAGG ACCGACACAG CCAGCCCGAT TC - #AGAGCCAC     13680     - AGAGTAATGG GACCAGATGA TCTTCACAGA CTCCCTTTCT CCCATAGATC TT - #GCACACCA     13740     - TAGTGGAGAC TTCCCATGTA CATCTATGGT TTGCCACTTA CAGAGTTACT TG - #GAGCCAGC     13800     - TGAAGTTAGA GCTGGCTTCT CCCCTTTGAG TCTTCAATTC TGTGTTTATG TG - #CAGGCCCG     13860     - GGGACCATGC CAGGCTTCTA AGAAGGTCTT CGAATGAAAG TCTGCTTGGG CT - #CTAGTGTG     13920     - TCCAGATCTC AGTGCCACTA TTATCCACTG ATATTGATCA AGTGCTGCTC TC - #CAGGAAGA     13980     - CCCCTGAGGT TTCCTGGTCC ATTGCAATGC ATGCTGGGTA CTCTTGCACT TG - #GATGGAAG     14040     - TAAAATCTCC TCACTAAACT CTGTGCCACC AAAATCTCCT TCTCAGTGTG AA - #TTGAAGAA     14100     - ACATTTTCCA AGACTTGCAT GTGCCAGGAG CCAAGGACTC AGAGTGATAA AA - #CAGCCTTC     14160     - TGCCCTCAGA GCTCTCTGTG GTGGGGCGCT TCCTGTGCTG TCTGGCTTTA CA - #CACAGCAG     14220     - GCAGAATGAC TTGAATTCGG CTGCTGTGCT GTCGTACAAC ATGCTGTTTA GG - #ATCTTGCA     14280     - CATGATAGCT AGGTATTCTT GCTTCAAATC GCAGGCACCC CACTTACCAA CT - #GTGTAGAC     14340     - TTGATCACGT TATTCAACCC CTGTGTCTCT GCTTCCTCAT TTTACAAATG GG - #GAGAAAAA     14400     - TAGCATCTAT CTCAAAGTTG TGAAAATTAA GCAAGTTAAT ACATATGTGC TA - #CGTAGAAC     14460     - AGTGCCTGGT ACATGGTCAG TTTTTGATAC ATGTTAGGTA TTATCATTAT TA - #TCACCTCC     14520     - AGAAACAATT TAAACTTCTC ATATAAGGCT CTCCAGACAC CTCTCATTGT CT - #TCCCTTCC     14580     - AAATCTGCAT TTATCTCTCT CTCTTTGCAG TCCAGTGTGA GGCTTGAATC AC - #CTATCAAG     14640     - CCTCACCTCC ACCCCTGTGC TTTACAAAAT GTCCTAGAGC TTCTATTTAC TC - #GTCTCACT     14700     - GCTCTGTGGG CTTTTTCACT CAAGGGCGTT TGCATGCTAT CCATTGCTAC CT - #GTTTTCTG     14760     - TTGCTGGTGT CTGTCTCCTG CTCTATCTTT GAAGAAAAGA AACAAGAAAA GG - #AATAACTG     14820     - AGAAACAGAG AAAAAAAATG TCTCTCCCTT CTGGTTCTTC CAGACCACCC AC - #TCATCCAT     14880     - CTTGTTCAAT GACAGCTTCT CTTCCTTTAA TTAATCACTG TGGTATATTT AT - #AAAGCTTA     14940     - TATTTATGAA AGACCTTTTA ATTTTTTAGT TATTAAAGCC CTTTCTCTTT GT - #CAGGTTGT     15000     - AACTGAGTGA GCTCTGGAGT TTGGAAAGAA GATCTTAGAA ATGGGCCAGA GA - #GCTCCTTC     15060     - TGAGATCCAA GCACGGAGAA TTGCACCTGC TGTGCATGGT AAGAGAGTGT GC - #TTGGTAGC     15120     - TCACAAGGGC AAGGTGAGAA TAGAAACTTT CATGCCTTTT TGATGGGGGT TA - #TGAAATCC     15180     - TACCAAGAAA CACCAGGTAT CAGATGTGGG GTCCTGTTTT CCCAAAGCCA CA - #AATGCTTG     15240     - AAGGAAGATC TTGTGTGATA AAATAATTAC CACATGAACC AATCTTGCAT GC - #ACAGCAAT     15300     - TTTGAGAGCC CATCCTGGGA GCTAGGTGTG TAGTGTTTAT CGTATTGTTG AG - #GCTCGTAA     15360     - AAATCTTGTA TGGCTGCAGG CAAGCCAAAC CCTTGACAGG CACTGCATCT CC - #GCTGACTC     15420     - TAGAAGACCA AGCCCAATTT CTTCCCTGTA TATAAGGGGA AGTCTCTATG CT - #TGGGGTAG     15480     - AGGAGTGTTT AGCTCCTTCC CTTACTCTAC CTTGCTCCTA CTTTTCTCTA AG - #TCAACATG     15540     - AGTCGACAGT TTAGTTCCAG GTCTGGGTAC CGAAGTGGAG GGGGCTTCAG CT - #CTGGCTCT     15600     - GCTGGGATCA TCAACTACCA GCGCAGGACC ACCAGCAGCT CCACACGCCG CA - #GTGGAGGA     15660     - GGTGGTGGGA GATTTTCAAG CTGTGGTGGT GGTGGTGGTA GCTTTGGTGC TG - #GTGGTGGA     15720     - TTTGGAAGTC GGAGTCTTGT TAACCTTGGT GGCAGTAAAA GCATCTCCAT AA - #GTGTGGCT     15780     - AGAGGAGGTG GACGTGGTAG TGGCTTTGGT GGTGGTTATG GTGGTGGTGG CT - #TTGGTGGT     15840     - GGTGGCTTTG GTGGTGGTGG CTTTGGTGGA GGTGGCATTG GGGGTGGTGG CT - #TTGGTGGT     15900     - TTTGGCAGTG GTGGTGGTGG TTTTGGTGGA GGTGGCTTTG GGGGTGGTGG AT - #ATGGGGGT     15960     - GGTTATGGTC CTGTCTGCCC TCCTGGTGGC ATACAAGAAG TCACTATCAA CC - #AGAGCCTT     16020     - CTTCAGCCCC TCAATGTGGA GATTGACCCT GAGATCCAAA AGGTGAAGTC TC - #GAGAAAGG     16080     - GAGCAAATCA AGTCACTCAA CAACCAATTT GCCTCCTTCA TTGACAAGGT GA - #GTTTCTCT     16140     - CTCATTGCAC TGGTAGGGCT GCCGCTGGTC CACTTGGGAT TGGTGCAGTC AA - #AACACATG     16200     - TAGGTTTGAA CCTCAAGTTT CCATGTTTAC ATGATTAAAA GGATGTTTTG TG - #GAATGGTC     16260     - TCCTAGGAGA TATGTTAGAT GTATGCTTGT GAATGGTGTT AATGACTCTC TC - #TTTGACAA     16320     - AGGGTTCGTG GTCGACCTAA AGGTGGGTCA GTGTGACATT AACATTTAAG TG - #CTTTTTAT     16380     - TCAGCTCTTG AGCGGAATTG GGACTCATAT CTGTTGAATG AAGATAATAG AA - #ATGGGGCT     16440     - AACTGAACTT TCCAGGGTGC AAGTGAGAAC CCTGGAAAGG TCTTCCTAAC CA - #TAGAAAGG     16500     - GAGTTGAGTG TGAACATAGT ATAGAGTGTT ATTGTAGCAG AAAACATGTG GT - #CAGTCAGT     16560     - GCCAAACATC TTTTGCTGTC AGAGGGGAGC TCTGCCTTCT AATAATTTTA CA - #TTGGTACT     16620     - GGATGAGGCT AGAGTTTTTT TATACTAATA TCTCCAAAAA TCAGCTCTAA AA - #AACTCAGA     16680     - TAAACCATTT TTTTAATTTT TTGCTTAATC ATTAATAGTG CCAATCCAAG GT - #TATCCACA     16740     - ACAAATTTCA AATCCAATTT TGAATTTTCC TGATATACTT TTGAAATGTG TG - #TGTGTCCT     16800     - GGGGATGCAA ACCAGTTTTT ATGGTAATAT ACCTAACAAA ATTTTGGAAG GC - #AAATCTCT     16860     - TAAATACCAT GCACCTATTT CAAAACATAA TTGCAATAAT TCTGTATGCG CT - #TTGCTATT     16920     - GGTATTTGTT TAGTTACTCC CTTCCAAGCC CTCTCTGAAT TAACAAGTTG GG - #TTTTATTA     16980     - TGCAGATGAT ATTAACTTGA TCATCTTCTT CCTATTTCTC TGTCATGGTC AG - #AAGATAGG     17040     - AATTGAGGTT CTTTTCCAAA TGAGGCACAG TTCTCCATGG CTATGAGACT CC - #ATTTATGC     17100     - ATCAGGAGTA AAGGGGTCTT GTGTTTTTAG GTGAGGTTCC TGGAGCAGCA GA - #ACCAGGTA     17160     - CTGCAAACAA AATGGGAGCT GCTGCAGCAG GTAGATACCT CCACTAGAAC CC - #ATAATTTA     17220     - GAGCCCTACT TTGAGTCATT CATCAACAAT CTCCGAAGGA GAGTGGACCA AC - #TGAAGAGT     17280     - GATCAATCTC GGTTGGATTC GGAACTGAAG AACATGCAGG ACATGGTGGA GG - #ATTACCGG     17340     - AACAAGTAAG GGACCCTGTC TGGGCAGTTC TTAACTTTTG CTGTAAAAGA GT - #TCCAGAAA     17400     - GTAATAAGCT AAGATCATGA AGCAGCATGT AGCTATGTCT TTTCTTAGGT TA - #GAGGCACA     17460     - TCAGTTTGAC ATTTTCAGAA ATCTTCATTT TCTCAGGAGA TGGAAATAGT CT - #AGTGGTTT     17520     - TATTGCTCAG TAGAAAGTAG TGGCCAATAT GTCCTAGGTT CATAATAGAA AG - #GCAGTGAT     17580     - AGGCAATGCC ACCTTTAGTT TAGAATGCTG GACTTCAGGT CTTACCACCT CT - #GAATCTCC     17640     - TAATTGTTTC TGCTTTCCTG CAGGTATGAG GATGAAATCA ACAAGCGGAC AA - #ATGCAGAG     17700     - AATGAATTTG TGACCATCAA GAAGGTAAGC AAATTCTGTA GGACGGAACT CA - #CATTTGAA     17760     - ATAAATAAGG GAAGAGGGTC TCCAATTACT AAGCAGAAAG CAGCCATGAT AT - #GGAGAGCC     17820     - AGGTAGTAGA CCTGGGGAGT ATATGGAGTG GGGCTATATT TTTCACATCA TC - #ATGGACCT     17880     - GGACTGATCC AGGCACTTGG CTTCTCCATA TTTCCCAGCA CCTTACATAG TA - #AGTGGAGT     17940     - GGCAGATTCT CAGCAAGCCA GGCACACTCC CTTGATGGTG CTATCCGGGG GT - #GGGACAGT     18000     - TAGGGAACTG TGATTTACCT GGGGCAAAAA GGAGTGGAGT AGACCCAAAG CT - #CCTTTTTT     18060     - TGCTTGGAGA ATCCCCTCAC AGGTAATGAG AGGGACCTGC CCTGGAGAGA AC - #GTGCCTTC     18120     - ATGATGTCCC TTGTTCCTCT AGGATGTGGA TGGTGCTTAT ATGACCAAGG TG - #GACCTTCA     18180     - GGCCAAACTT GACAACTTGC AGCAGGAAAT TGATTTCCTT ACAGCACTCT AC - #CAAGCAGT     18240     - AAGTCTTCCA GTTTCAACCA AGTTTATCTA AATGGAGAGT TTTTAAGCCG GA - #ACCCACAA     18300     - CGATTCAGAA GAATAGATAT TTATCTTTTA TTTCCTGACT GCTTTCTCTG TC - #TAAGTTGT     18360     - TTTTTGTTTT AGTGCTGTAA GAGTCACTAA CCTATTATGT CTTGCAGGAG TT - #GTCTCAGA     18420     - TGCAGACTCA AATCAGTGAA ACTAATGTCA TCCTCTCTAT GGACAACAAC CG - #CAGTCTCG     18480     - ACCTGGACAG CATCATTGCT GAGGTCAAGG CCCAGTACGA GGATATAGCC CA - #GAAGAGCA     18540     - AAGCTGAGGC CGAGTCCTTG TACCAGAGCA AGGTGAGTGG GCTGAAACCG TA - #GCCAGTTT     18600     - CCCTGAAATG GCTTGTCTTG CTATCCTGTG TTATCTCATG TATGTGTGCC TG - #TGCCATGC     18660     - TGAGTTCTGC CTACATTTAA CAAACGCTAT CTACCATCTT TAGTATGAAG AG - #CTGCAGAT     18720     - CACTGCTGGC AGACATGGGG ATAGTGTGAG AAATTCAAAG ATAGAAATTT CT - #GAGCTGAA     18780     - TCGTGTGATC CAGAGACTTA GATCTGAAAT CGACAATGTC AAGAAGCAGG TA - #TGTGCTTT     18840     - CTCCTTCTAC CACTCAGCTG TATGGAATGG GGGTAACCCT CAGGTAAAGG GC - #GAGTGCTT     18900     - TCCTAGTTTT GAATCTTGCA ATTCAGCCCA AGGCTACATT ATTAGCCCTG GT - #TCCTTTTC     18960     - TGACTATGCT AGTTTCCAGA ATGCAGCCAT CATGCTGGGT TCTCTTTAGG GA - #AATCTGTG     19020     - AGAATGGCCT AGTAGAGAAA GATGGGATGG TCAATGTGAG TGATCTAGCC TA - #TGACCCAA     19080     - AGTGGACTTA AGAGTTGGGG AGTGAGAGGA AGGGCAGCCA GGAGGTTTTA GA - #GTAGGTGT     19140     - TTAGAAGAAT GTCAAGTCTG TAAGGGTTGT AGGAGCCTTG ACTCAGGGCC AA - #GAGAGGCT     19200     - GTTGAGTTAT CCCTAAGGTC TTTTAAGGAA GTCAACATGG TGATGTGTTA TC - #TGGAGGTG     19260     - GGTGTGAGAT GACTTAAGGC CAAGTGGTTC TGTTGGACTC ATTATTGGCC TC - #ACTGGAGT     19320     - GGGGAGACCA ATTGGGATGA GGAGGCCTAG TGGGGAATGC ATATTATGAG AG - #GGTGTCAT     19380     - ATCTTTTTCA GATCTCCAAC TTGCAGCAGT CCATCAGTGA TGCAGAGCAG CG - #TGGCGAGA     19440     - ATGCCCTCAA GGATGCCAAG AACAAGCTGA ATGACCTGGA GGATGCCCTG CA - #GCAGGCCA     19500     - AGGAAGACCT GGCCCGCCTG CTGCGTGACT ACCAGGAGCT GATGAACACC AA - #GCTGGCCC     19560     - TGGATCTGGA GATTGCCACC TACAGGACCC TCCTGGAGGG AGAAGAAAGC AG - #GTGAGGAA     19620     - GGGACGCTGG GAGTCGAACC TCTTCTCATG GTCTTCCTTC CTTGCAAGCT GA - #TTGTTGTT     19680     - GAAGATGCAG CCATCTGATT GCAGCTTGTG CTGGGTATGG GGAAATGAAA AG - #TACACGGA     19740     - GCAGGAGGAA GGACCTAGTT TTACTTTGGG AGCTGGAGTC CCAAGCTGTT TA - #TTTTTTTC     19800     - TTCTAGGGCT GTAACATATC TAGAAAGAGC TTTGAGGTGG AGCAAATTAT TC - #TTTATCTG     19860     - GGCTGCCTCA GATGGCAGCT GGCCTAAAGT CGGCATCTTT AGAGGGGGCC TT - #CATTGGCT     19920     - GCAAGGCTCG TCTCGTTTAT ATGGGAATTT CTCCGTGTTT GTACTCTTGC TG - #AGAAAAAA     19980     - TGACAGGTCT GGGAGGCCAG AGGGGATTGG ATTAAGTTTC AGATTAAGTG CA - #TTGGAGAA     20040     - GACCCAGATG GGGAAAGTCT TCAAGGTGGT GGAGCGGGGA ATGGGGAAGC GG - #TTTGGGAA     20100     - GCTGGAGTGT CCTGAGGAAT TTTCTTATTT TCTCCTACAG GATGTCTGGA GA - #ATGTGCCC     20160     - CGAACGTGAG TGTGTGTAAG TACAAGTCGA TTTCTCAGGG GCATGTGCAG GC - #TTTGTTGG     20220     - GCTGGAAACG GAGTTGAGGT TGAAAATAAC TGAGCTTCCT CTTGCAGCTG TG - #AGCACAAG     20280     - CCACACCACC ATCAGTGGAG GTGGCAGCCG AGGAGGTGGC GGCGGTGGCT AC - #GGCTCTGG     20340     - AGGTAGCAGC TATGGCTCCG GAGGTGGTAG CTATGGTTCT GGAGGTGGCG GC - #GGCGGCGG     20400     - CCGTGGCAGC TATGGCTCCG GAGGTGGCAG CTATGGCTCT GGAGGTGGCG GC - #GGCGGCCA     20460     - TGGCAGCTAC GGCTCCGGAA GCAGCAGTGG GGGCTACAGA GGTGGCTCTG GA - #GGCGGCGG     20520     - CGGCGGCAGC TCTGGCGGCC GGGGCTCTGG CGGCGGGAGC TCTGGAGGCT CC - #ATAGGAGG     20580     - CCGGGGATCC AGCTCTGGGG GTGTCAAGTC CTCTGGTGGC AGTTCCAGCG TG - #AAGTTTGT     20640     - TTCTACCACT TATTCCGGAG TAACCAGATA AAGAGATGCC CTCTGTTTCA TT - #AGCTCTAG     20700     - TTCTCCCCCA GCATCACTAA CAAATATGCT TGGCAAGACC GAGGTCGATT TG - #TCCCAGCC     20760     - TTACCGGAGA AAAGAGCTAT GGTTAGTTAC ACTAGCTCAT CCTATTCCCC CA - #GCTCTTTC     20820     - TTTTCTGCTG TTTCCCAATG AAGTTTTCAG ATCAGTGGCA ATCTCAGTCC CC - #TGGCTATG     20880     - ACCCTGCTTT GTTCTTTCCC TGAGAAACAG TTCAGCAGTG ACCACCACCC AC - #ATGACATT     20940     - TCAAAGCACC TCCTTAAGCC AGCCAGAGTA GGACCAGTTA GACCCAGGGT GT - #GGACAGCT     21000     - CCTTAGCATC TTATCTCTGT GCTGTTTTGG TTTTGTACAT AAGGTGTAAG CA - #AGTTGTTT     21060     - TTCTTTTGTG GAGAGGTCTT AAACTCCCCA TTTCCTTGTT TTGCTGCAAT AA - #ACTGCATT     21120     - TGAAATTCTC CATGTCTCGA TCGCCCTTGT TTACGGCACT GTCTAACCTG GA - #TGGGTGTT     21180     - TTGTGAGGTA AAAGAAGACA CTAGAGCCAC ATGGCATATG GGAAAGTCAT GC - #ACACAAAC     21240     - ATGAGAAAAA TGCAGAGGCC AACCAGGCAA CATTTCACCA GACTGGAATC AC - #AGAGAGAG     21300     - CAAGCACTTT CCCAGATGGT GGGGATGTCA TGGAGAAATG GAGAGACCGG GT - #GACAGGTT     21360     - TTGTTCATTT GAGAAGGCTT TCTTGAAAAG GGCAGTGAGC AAGCAGGTTG GG - #AGGAAGAG     21420     - GTGTGGCATT GAGAAGAAGG GAAAGTATTG CATGAAAAAG TAATTCTTCA CG - #TGGAACAG     21480     - CCAGTAAGGA GGGGCATGAG TAATATAGGG TCAGCAGTTA CTGGAGCCAG AA - #TACAGACT     21540     - TTGGCCTGGG GAGTTCAAGA ACTAAGAGTG GTAATAGAGA GTTGGATATT CC - #ATTTCCCT     21600     - TCTCTTTTTG TGCCACCACC CAAAGCTCTG CATAATCTAA GAAGTTCCCT TG - #TTGACACA     21660     - TAGCTCATAC TTGTGAAGTT GTACAACAGG ATAGCATAGT GGCCAGAAGC AT - #GGACAGTT     21720     - GAACTCAGAT ATGCTTGGGT TTGAATCTTA CCATCACCAT TTACTAGTTC TG - #TAATACAG     21780     - TGCAAGTTAC AGACATCTCT GCACCTCAGT TTTAGTATGT CTAAATTGGG GA - #TGATAATG     21840     - CCTTCCTTGT GGGGATAGTG TGAGGATTGA ATAAGATGAA TACACATGGC TG - #AGCACACA     21900     - GCAAGCACTA AATAAGTGCC AGTTTTAATG ATAACGGTGA TGATGATGAT GA - #TGATGATG     21960     - ATGACGTAAC ATTGCTTGTG GGACTCCATA CAGCTCAGTA GATGCTTGCT CA - #AAGAAGCA     22020     - AGTTACCAAA ATTTTTGTAA TGGTTCTATG AACGTGAAAA AAGCAGTCAA CT - #TCTCTGAG     22080     - GATCAATTTC CTTAGTTTCC AATTAGGAAA AGTCTTCTTA GCTCCAGAGT CC - #CACAGGGC     22140     - TAATGGAATA AGGAGAGGAT AGATCACACA TGTATTATGC AAACACAACT CA - #GGTGAGCT     22200     - CTATTCTTCC TTCTCAGTTA TCCCTTCTGT AGGGACCCCA GTGTCCCCTG CT - #GTCTTTCT     22260     - GTGTCCTGAC CGGGAAACAC AGTGTGCCTT GTCTACTCCA TCACTTGGCC AG - #CTGCATGC     22320     - TTTCCTTTGC AGGCTTGAAG CAAAGCTGGG TCTCGGACAT TCTCAGGCAC TG - #ACAAAGCT     22380     - GTTTAGTTGT TGCTGGGAAA CACTGGGAAA TAGCCCTTTT GTTAAACACA CA - #GAAACTAG     22440     - CCTTCGCCCT GAGCCAAATT CCTTAAACTC GTCTATGAAA TTCCATAACC TG - #ACTCCTTA     22500     - ACTGCAGACA TACCCAGCTA GAACATCCCT CATGTCCCTG TCCACCGTGA GA - #ATGCTGCA     22560     - CTTCACTCTG AACCTTTAGT CCTCCTTTTA AATACTGCAC ACTGATCACC CT - #GGTGTTTA     22620     - GTGCTTTGTT TTTTGGAATC CCACCTGGCT CCATTTTGGG ATGGTTCCGG GC - #ACTTCCCT     22680     - ATGGAAATTC CCCTGCTGTC ACTGTCAGAG TGAGTCCAGC AGTGGGTTTA GC - #TGGATGAA     22740     - ACACCACCAT GTCCATTTCC ATTCAGACTA ATGTCAGAAT TTGAAAGGCA CT - #ATGGTAGA     22800     - GTAGAAAGAA CAAGGAACTG TACTATTTAA AGGGCAGGCA AAGAAAAGGC AT - #CTATAGCT     22860     - TATAAGATGT GTGGATCTTT GGATGTGACT TGGCCATCCT GAGCCTAAGT TG - #TCTTGTAG     22920     - GAGAAATGGG AATGAGAATA TTTTCCTCTA GACATCAAGA GGAAAAGAAA TA - #TAACGTGA     22980     - AAACCTTTGT GAATTGTGAA TGTGTTATAC AGAGTAGCTA AAAGAATTAA AA - #AGGGAGTG     23040     - ACAAAAAAGT AAAAGGCAGC TGGCTGCTCA GGGCCTCCAT GGAGGGAAGT AC - #CTTGATAT     23100     - GGTCACTGTG GCTCAGTGAC AGCTCTGCAG GGACAGGAAA TTGATTTGTT AG - #TGCACCCA     23160     - AAGTTGAATC TGCTCCTGAG TACTGATTTA TGGGAACCAA ACACACAAGA GA - #TGAAGGAT     23220     - GTGTCAACCA GAATGTCCAG CATTAGCTTG TGGGGAAACA CATACTTCCA GT - #GACTGAAA     23280     - TACCATCCTG TTATCAAGAG ATCTGGGAAA CTAAAGTACT GACAAGAGCT GG - #CTTGATCT     23340     - GTGGATTTAG AACAATGAGA GTTAGGTGGC CTTGAGGGAG ATGATTCACT CT - #CCTTCACA     23400     - GAAGAGCTGA CCTCTGGGGT CAACAGATAT AGCACCTCTT TCCCAGGGAC GC - #TACTGAAT     23460     - GAACAGTGAT GTGTTCTTAT ACTCTGGCCC AGATTTTCTA CATACTTTCT TA - #GGTTACAA     23520     - CTTTATTTAG TCACATTTCA GTACTGGGGA TACTCCTGTT TATCTTCTTT GG - #ACTCGAGT     23580     - TTTTATGGGA AGGTCATGAA ACAGAGAAAA ATACAATTTG CAGGGAAACT TA - #CCAAGGCT     23640     - TGTAAGGTTA CAAGGATTAA ATGAAAACCC TGTGTAAGTC AGTATATAGT GA - #AGAAGTAA     23700     - ATTGAGTTAG ACCAAACGCC AAAATGCATC CGCATTAGAA AGACGATAAA GG - #AAGACTCT     23760     - GGATTCAGTT CTGTTCAAAA AACATTTTCT GCACAAATAC TATGTATGAG GA - #ACTGGGCG     23820     - TTGGGGAGAT GATGATGAGT GAGACATGGT TCTTGCTTTC AGAGAGCCTA GA - #GACCTGGG     23880     - TGGTAGCAAT GGTAGAGATA CATCCAAGAC ACAGAAATAG ATATACAGGA AC - #ACAGATGA     23940     - TTGAAAGTGA TGCTTGGCAG GGCTTTAAAG AATGAATCAG AGTTTTTCAG GC - #AGACGAGG     24000     - ATCTTCAAGG CAGAGGGAAT CATATAGATA AGGACATAGA AGAGTGAAAT TT - #CATGAAGT     24060     - AGTTAAGCAT CTGAAGAAGC ATGGAATTAG TGACAAGAAA TGATGCGGAA AA - #GATATCCA     24120     - GATCCAATCA AGAAGGGCCT TGTTGGCATT CTATGGAGTC TGGACTTTGG CT - #TCTGGGTC     24180     - ACAAGTTCTC AGATGGGGTT TTCATATCTA TTATTAGACC TACTATGTAC TG - #GTCCAGTG     24240     - GAAGGGAAAG GGGTTGTCTT ACTGCTAGTG GAGTAGGAAT TGGGTATGGA CC - #ACAGCTTG     24300     - TCTTGTTTCC AAGTATTCCC TAAGAAATCT GGTCTGCTGA TGGGAGATCT AT - #TTATGGAA     24360     - ATGTCTTTTT CCCTCAGGAA TTTTATGTCG GAAACAGCTG TCATAGGTGA GG - #AGGAACTG     24420     - GTAAAAGTAC TTAATAGGAG AGTGTCATGG TCAGATTGGT GTTTTGGAAA AG - #TCAGCCAG     24480     - GGCAGATTGG AGAGGTCCAT ATTGGAGGCA GGAAGACTTA AGAGACTATT GC - #AAAGGTGA     24540     - AGACAAAAGA CGATAGGGAC TTGCACTTTA ATTCCAGCCC TTAGAAGTAG TA - #GAAGGTCA     24600     - GAAATGAGAA TATGCATTAC AGAGATAGTT AGTTGCTATA TCATTAGGAC TT - #GGTGATAG     24660     - ATTGGATGAG GATGCGGTTG GGTGAGGCAA AGAGGAGAGT CCACATTCCT GG - #TCTGGGTA     24720     - GTAACAAAGA ATCTAGCAAG AGGGCTTGTG GGGAAAGATG CTGAGTTACG TA - #GCAAGTGC     24780     - ATCTGCTTTA TCCTTGTAAT GAATGGGGCT AAAGGTGTAA ACCAAAGAGT CA - #TCAGCATT     24840     - TGGAGGGTAG AATAAATCAT CAGATAACTC AGGAAGAAGG AGCAGAAGAA TT - #ACTGATAC     24900     - TCCCTGGAAG GAAAACCGGA AGTAAATGGG AGAAACTTGC TCAAGTGGAC AA - #AGTTTAAC     24960     #               24979C     - (2) INFORMATION FOR SEQ ID NO:  4:     -      (i) SEQUENCE CHARACTERISTICS:     #            8 amino acids     #              amino acid     #        singleTRANDEDNESS:     #           linearLOGY:     #       peptideCULE TYPE:     -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  4:     #Arg Lys Ser Tyr Lys His     #            5 1     - (2) INFORMATION FOR SEQ ID NO:  5:     -      (i) SEQUENCE CHARACTERISTICS:     #            16 amino acids     #              amino acid     #        singleTRANDEDNESS:     #           linearLOGY:     #       peptideCULE TYPE:     -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #  5:     - Met Ser Ser Val Lys Phe Val Ser Thr Thr Ty - #r Ser Gly Val Thr Arg     #                15     __________________________________________________________________________ 

We claim:
 1. A vector for expression of a nucleic acid sequence in an epidermal cell, wherein said nucleic acid sequence is other than a mammalian K1 keratin gene, said vector comprising, in operable association:a 5' flanking region from a mammalian gene including necessary sequences for expression of said nucleic acid sequence; a 3' flanking region from a mammalian K1 keratin gene which regulates expression predominantly in an epidermal cell of said nucleic acid sequence with which it is associated; and a linker connecting said 5' flanking region to said nucleic acid sequence, said linker having a position for inserting said nucleic acid sequence, wherein said linker lacks the coding sequences of a gene with which it is naturally associated.
 2. The vector of claim 1, wherein said 3' flanking region is 3' to said position for inserting said nucleic acid sequence.
 3. The vector of claim 1, wherein said 3' flanking region is 5' to said linker.
 4. The vector of claim 1, wherein said vector further comprises a nucleic acid sequence.
 5. The vector of claim 1 or 4, wherein said 5' flanking region includes a promoter, 5' UTR, and a first intron and intron/exon boundary in appropriate relationship for expression of said nucleic acid sequence.
 6. The vector of claim 1 or 4, wherein said 3' flanking region includes a 3' UTR and a 3' NCR containing a transcriptional termination region.
 7. The vector of claim 1 or 4, wherein said 3' flanking region is from a human keratin K1 gene.
 8. The vector of claim 1 or 4, wherein said 5' flanking region is from a keratin K1 gene.
 9. The vector of claim 5, wherein said 5' flanking region is approximately 1.2 kb, said first intron and intron/exon boundary is 1.0 kb, and said 3' flanking region is approximately 3.9 kb of a keratin K1 gene.
 10. The vector of claim 5, wherein said 5' flanking region is approximately 1.2 kb, said first intron and intron/exon boundary is 1.0 kb, and said 3' flanking region is approximately 4.3 kb of a keratin K1 gene.
 11. The vector of claim 1 or 4, wherein said 5' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;said 3' flanking region comprises nucleotides 6891 to 10747 of Sequence ID No. 1; and said linker comprising nucleotide 2351 to 2376 of Sequence ID No.
 2. 12. The vector of claim 1 or 4, wherein said 5' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;said 3' flanking region comprises nucleotides 7895 to 7921 of Sequence ID No. 1; and said linker comprising nucleotide 2351 to 2376 of Sequence ID No.
 2. 13. The vector of claim 1 or 4, wherein said 5' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;said 3' flanking region comprises nucleotides 7924 to 7948 of Sequence ID No. 1; and said linker comprising nucleotide 2351 to 2376 of Sequence ID No.
 2. 14. The vector of claim 4, wherein said nucleic acid sequence comprises genetic material coding for a protein, or a polypeptide.
 15. The vector of claim 14, wherein said genetic material codes for a hormone, a growth factor, an enzyme, a drug, a tumor suppressor, a receptor, an apolipoprotein, a clotting factor, an antigen, an oncogene or a transforming gene.
 16. The vector of claim 15, wherein said oncogene is selected from the group consisting of ras, fos, myc, erb, src, sis or jun.
 17. The vector of claim 14, wherein said genetic material of said nucleic acid sequence contains the E6 or E7 transforming sequence of human papilloma virus.
 18. The vector of claim 14, wherein said genetic material of said nucleic acid sequence encodes for a protein or polypeptide selected from the group consisting of a TGF-α sequence, proinsulin, insulin, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding protein, epidermal growth factor, dermal growth factor, an angiogenesis growth factor Type IV collagen, laminin, nidogen, Type VII collagen, TGF-β, acidic fibroblast growth factor, angiogenin, cytokine receptor, and IL-1, IL-6 and IL-8, a viral capsid protein.
 19. The vector of claim 1 or 4, wherein said vector further comprises a modulator sequence associated with said 3' flanking region or said 5' flanking region.
 20. The vector of claim 19, wherein said modulator sequence comprises sequences responsive to calcium, Vitamin D or its metabolite, Vitamin A or its metabolite, or progesterone.
 21. An isolated or cultured mammalian epidermal cell transformed with a vector for expression of a nucleic acid sequence, wherein said nucleic acid sequence is other than a mammalian K1 keratin gene, said vector comprising, in operable association:a 5' flanking region from a mammalian gene including necessary sequences for expression of said nucleic acid sequence; a 3' flanking region from a mammalian K1 keratin gene which regulates expression predominantly in an epidermal cell of said nucleic acid sequence with which it is associated; and a linker connecting said 5' flanking region to said nucleic acid sequence, said linker having a position for inserting said nucleic acid sequence, wherein said linker lacks the coding sequences of a gene with which it is naturally associated.
 22. The epidermal cell of claim 21, wherein said 5' flanking region includes a promoter, 5' UTR, and a first intron and intron/exon boundary in appropriate relationship for expression of said nucleic acid sequence.
 23. The epidermal cell in claim 21, wherein said 5' flanking region is approximately 1.2 kb, said first intron and intron/exon boundary is 1.0 kb, and said 3' flanking region is approximately 3.9 kb of a keratin K1 gene.
 24. The epidermal cell of claim 21, wherein said 5' flanking region is approximately 1.2 kb, said first intron and intron/exon boundary is 1.0 kb, and said 3' flanking region is approximately 4.3 kb of a keratin K1 gene.
 25. The epidermal cell of claim 21, wherein said nucleic acid sequence comprises genetic material coding for a protein, or a polypeptide.
 26. The epidermal cell of claim 25, wherein said genetic material codes for a hormone, a growth factor, an enzyme, a drug, a tumor suppressor, a receptor, an apolipoprotein, and an antigen.
 27. The epidermal cell of claim 21, wherein said vector further comprises a modulator sequence in said 5' flanking region or said 3' flanking region.
 28. The epidermal cell of claim 21, wherein said 3' flanking region includes a 3' UTR and a 3' NCR containing a transcriptional termination region.
 29. The epidermal cell of claim 22, wherein said 5' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;said 3' flanking region comprises nucleotides 6891 to 10747 of Sequence ID No. 1; and said linker comprising nucleotide 2351 to 2376 of Sequence ID No.
 2. 30. The epidermal cell of claim 22, wherein said 5' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;said 3' flanking region comprises nucleotides 7895 to 7921 of Sequence ID No. 1; and said linker comprising nucleotide 2351 to 2376 of Sequence ID No.
 2. 31. The epidermal cell of claim 22, wherein said 5' flanking region comprises nucleotides 1 to 46 of Sequence ID No. 1;said 3' flanking region comprises nucleotides 7924 to 7948 of Sequence ID No. 1; and said linker comprising nucleotide 2351 to 2376 of Sequence ID No.
 2. 32. The epidermal cell of claim 27, wherein said modulator sequence is responsive to calcium, vitamin D or its metabolite, vitamin A or its metabolite, or progesterone.
 33. The epidermal cell of claim 27, wherein said 3' flanking region further includes an 18 KB EcoRV fragment.
 34. The epidermal cell of claim 25, wherein said nucleic acid sequence encodes for proinsulin, insulin, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding protein, epidermal growth factor, dermal growth factor, an angiogenesis growth factor Type IV collagen, laminin, nidogen, Type VII collagen, TGF-β, acidic fibroblast growth factor, angiogenin, cytokine receptor, and IL-1, IL-6 and IL-8, a viral capsid protein. 